CA2268457A1 - Mammalian rad1 genes, polypeptides and methods of use - Google Patents

Abstract

The present invention provides isolated nucleic acid molecules encoding mammalian RAD1p polypeptides (such as human and marine RAD1p polypeptides) and allelic variants, mutants, fragments or derivatives thereof.
The invention also provides mammalian RAD1p polypeptides encoded by such isolated nucleic acid molecules, antibodies binding to such polypeptides, genetic constructs comprising such nucleic acid molecules, prokaryotic or eukaryotic host cells comprising such genetic constructs, and methods and compositions for use in diagnosing and treating disorders characterized by, or utilizing, a loss of cell cycle checkpoint control or by increased sensitivity to DNA-damaging agents.

Description

Mammalian RADI .Genes, Polypeptides And Methods of Use BACKGROUND OF TFiE INVENTION
Field of the Invention The present invention is in the fields of molecular biology, medical diagnostics and therapeutics. In particul~~r, the invention provides isolated nucleic acid molecules encoding mammalian. homologues ofthe yeast radl + gene (i.e., mammalian RADl genes), such as human RADI (HRADI), marine RADI
(MRA.Dl ), and mutants, fragments or derivatives thereof. The invention also relates to polypeptides encoded by such isolated nucleic acid molecules, antibodies binding to such polypeptides, I;enetic constructs comprising such nucleic acid molecules, prokaryotic or eukaryotic host cells or whole animals (including transgenic animals) comprising siuch genetic constructs, and methods and compositions for use in diagnosing disorders associated with sensitivity to DNA damage (e.g., cancer), and therapeutic methods for treating certain disorders (such as cancer) by inducing increased sensitivity to DNA damage in cells.
Related Art Cell cycle checkpoints are regulatory mechanisms which prevent cell cycle progression in the presence of DNA damage or incompletely replicated DNA (Weinert, T.A., and Hartwell, L.H., S~:ience 241:317-22 (1988); Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol. 10:6554-64 (1990); al-Khodairy, F., and Carr, A.M., EMBO J. 11:1343-SO (1992); Rowley, R., et al., EMBO J.
11:1335-42 (1992)). Even in the absence of exogenous DNA damage, defects in cell cycle checkpoints lead to genomic instability, as demonstrated in budding yeast rad9 mutants at the G2-M checkpoint (Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol. 10:6554-64 (1990)), and in mammalian p53 null cell lines at the G1-S checkpoint (Livingstone, L.R., et al.,Cell 70:923-35 (1992); Yin, Y., et al., Cell 70:937-48 ( 1992)). The resulting widespread genomic abnormalities are typical of cancer cells (Howell, P.C., Science' 194:23-28 ( 1976)). A large body of evidence implicates defects in cell cycle regulation, including checkpoint regulation, in the genesis of human cancers (Swift, M., et al., N. Engl. J.
Med.
316:1289-1294 (1987); Malkin, D., et al., Science 250:1233-8 (1990);
Srivastava, S., et al., Nature 348:747-9 (1990); Kastan, M.B., et al., Cancer Res. 51:6304-11 ( 1991 ); Motokura, T ., et al., Nature 350: S 12-5 ( 1991 ); Swi ft, M., et al., N.
Engl. J. Med. 325:1831-1836 (1991); Donehower, L.A., et al., Nature 356:215-21 (1992); Kastan, M.B., et al., Cell 71:587-97 (1992); Kuerbitz, S.J., et al., Proc.
Natl. Acad. Sci. USA 89:7491-5 (1992); Liviingstone, L.R., et al.,Cel170:923-(1992); Xiong, Y., et al., Cell 71:504-14 (15192); Yin, Y., et al., Cell 70:937-48 (1992); O'Connor, P.M., et al., Cancer Res. 53:4776-80 (1993); Serrano, M., et al., Nature 366:704-7 ( 1993 ); Dulic, V ., et al, Cell 76:1 O 13-23 ( 1994);
Kamb, A., et al., Science 264:436-40 (1994); Nelson, ~7V.G., and Kastan, M.B., Mol.
Cell.
Biol. 14:1815-23 (1994); Savitsky, K., et al., Science 268:1749-53 (1995)).
The increased genomic instability evident in all checkpoint mutants so far tested is a likely mechanism for increasing cancer susceptibility.
The fission yeast Schizosaccharomyces pombe undergoes a dose-dependent G2 delay in response to DNA damage caused by radiation (al-Khodairy, F., and Carr, A.M., EMBO J. 11:1343-SO (1992); Rowley, R., et al., EMBO J. 11:1335-42 (1992)). Cells remain arrested at this G2 checkpoint while DNA damage is repaired, then enter mitosis and resume progression through the cell cycle. Six "checkpoint ra~fi' genes have been identified in S.
pombe: radl+, rad3+, rad9+, radl7+, rad26~, and hulsl+ (al-Khodairy , F., and Carr, A.M., EMBOJ.11:1343-SO (1992); Enoch, T., et al., GenesDev. 6:2035-46 (1992); Rowley, R., et al., EMBO J. 11:1335-42 (1992); al-Khodairy, F., et al., Mol. Biol. Cell 5:147-60 ( 1994)). All six of these genes are required to arrest the cell cycle in response to either DNA damage or incomplete DNA replication.
Mutations in any of the checkpoint raf genes result in almost identical phenotypes; these mutants are all sensitive to treatment with DNA-damaging agents (e.g. , ultraviolet (UV) or y irradiation ) and/or agents that block replication (e.g., hydroxyurea). This sensitivity is dine to loss of the G2 and S phase checkpoints which normally prevent mitotic entry in the presence of damaged or incompletely replicated DNA, respectively (al-Khodairy, F., and Carr, A.M., EMBO J. 11:1343-50 (1992); Enoch, T. et al., Genes Dev. 6:2035-46 (1992);
Rowley, R., et al., EMBOJ:11:1335-42 (1992); al-Khodairy, F., et al., Mol.
Biol.
Cell. 5:147-60 (1994)). These data suggest that these genes share a common or overlapping pathway.
While the G2 checkpoint also exists :in human cells, little is known about the molecular mechanisms involved. Recent identification of human genes homologous to known G2 checkpoint control genes in yeast suggests that the regulation of this process has been conserved from yeast to man. For example, a functional homologue of the rad9+ gene, it~RAD9, and a structural homologue of the rad3+ gene, ATR, have previously been reported in humans (Bentley, N.J., et al., EMBOJ. 15:6641-6651 (1996); Cimprich, K.A., et al., Proc. Natl. Acad.
Sci. USA 93:2850-2855 (1996); Lieberman, H.B., et al., Proc. Natl. Acad. Sci.
USA 93:13890-13895 (1996); and WO 9T/46661 A2 (U.S. Application No.
08/644,034, filed May 9, 1996)). However, a mammalian structural homologue of the RAD1 family of checkpoint control genes (e.g., radl+ in fission yeast, RAD17 in budding yeast and RECI in Ustilago maydis) has not heretofore been reported.
SUMMARY OF THE INVENTION
The present invention generally :relates to mammalian cell cycle checkpoint control genes and polypeptides, and methods of diagnosing and treating certain mammalian disorders using these genes and polypeptides.
Specifically, the invention provides manunalian homologues of the yeast checkpoint control gene radl +, herein designated "mammalian RADI genes,"
such as isolated human or marine RADl (iYRADl and MRADI, respectively) nucleic acid molecules.

Isolated HRA.Dl nucleic acid molecules of the invention preferably comprise a polynucleotide having a nucleotide sequence at least about 65 %
(more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the g~~oup consisting of (a) the nucleotide sequence set i:orth in SEQ iD NO:1;
(b) a nucleotide sequence encodi ng the HRAD 1 p polypeptide having the complete amino acid sequence set forth in SEQ ID N0:2;
(c) a nucleotide sequence encoding the HRAD lp polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 011905;
(d) a nucleotide sequence of a polynucleotide which hybridizes under stringent conditions to a polynucleotide haviing the nucleotide sequence set forth in SEQ ID NO:1; and (e) a nucleotide sequence complementary to any one ofthe nucleotide sequences in (a), (b), (c) and (d), or a fragment thereof.
Analogously, isolated MRADl nucleic acid molecules of the invention preferably comprise a polynucleotide having a nucleotide sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at le~cst about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of (a) the nucleotide sequence set i:orth in SEQ ID N0:3;
(b) a nucleotide sequence encoding the MRAD 1 p polypeptide having the complete amino acid sequence set forth in SEQ ID N0:4;
(c) a nucleotide sequence encoding the MRAD lp polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 038841;
(d) a nucleotide sequence of a polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID N0:3; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d), or a fragment thereof.
The invention is also directed to an isolated nucleic acid molecule comprising a polynucleotide encoding an ep itope-bearing portion of a HRAD 1 p or MRADIp polypeptide, wherein the epitope-bearing portion is selected from the group consisting of a polypeptide having; an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID N0:2 (for HRADIp) or from about 77 to about 86 in SEQ ID N0:4 (for MRADIp);
a polypeptide having an amino acid sequence: consisting essentially of amino acid residues from about 89 to about 97 in SEQ II) N0:2 (for HRAD 1 p) or from about 89 to about 97 in SEQ ID N0:4 (for MR.AD 1 p); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in SEQ ID N0:2 (for HRADlp) or from about 112 to about 128 in SEQ ID N0:4 (for MRADIp); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in SEQ
ID N0:2 (for HRADlp) or from about 159 to about 177 in SEQ ID N0:4 (for MRADIp); and a polypeptide having are amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID N0:2 (for HRAD 1 p) or from about 227 to about 2;5 7 in SEQ ID N0:4 (for MRAD 1 p).
The present invention also relates to vectors, particularly expression vectors, which comprise the isolated nucleic acid molecules of the present invention, and to host cells comprising these; vectors. Preferred host cells of the invention include, but are not limited to, bacterial cells, yeast cells, animal cells (especially mammalian cells or insect cells) and plant cells. The invention also relates to methods of making such vectors and host cells, and methods of using the same for production of HRAD 1 p or MRAD 1 p polypeptides by recombinant techniques.
The invention also is directed to isolated HRADlp polypeptides and isolated MRAD lp polypeptides. Isolated HF;AD 1 p polypeptides preferably have an amino acid sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identiical to a reference sequence selected from the group consisting of (a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the HRAD lp polypeptide as set forth in SEQ ID N0:2; and (c) the complete amino acid sequence of the HRADIp polypeptide encoded by the cDNA clone having GexiBa~zlc Accession Number AF 011905, or a fragment thereof.
Analogously, isolated MRAD 1 p polypeptides of the invention preferably have an amino acid sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80°ro, at least about 85%, at least about 90%, at least about 95% or at least about 99'%) identical to a reference sequence selected from the group consisting of (a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID N0:3;
(b) the complete amino acid sequence of the MRADIp polypeptide as set forth in SEQ ID N0:4; and (c) the complete amino acid sequence of the MRADlp polypeptide encoded by the cDNA clone having GenBa~ik Accession Number AF 038841, or a fragment thereof.
The invention alsa relates to other isolated HRAD 1 p and MR.AD 1 p polypeptides (as defined below) comprising ane or more epitope-bearing portions of the above-described HRADlp and MItADIp polypeptides, wherein the epitope-bearing portion is selected from thc; group consisting of a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID N0:2 (for FIRADIp) or from about 77 to about 86 in SEQ ID N0:4 (for MRADIp); a ~polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about in SEQ ID N0:2 (for HRADlp) or from about 89 to about 97 in SEQ ID N0:4 (for MRADIp); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in SEQ ID N0:2 _7_ (for HRA.D lp) or from about 112 to about 1:Z8 in SEQ ID N0:4 (for MRADIp);
a polypeptide having an amino acid sequence; consisting essentially of amino acid residues from about 159 to about 177 in SE;Q ID N0:2 (for HRADIp) or from about 159 to about 177 in SEQ ID N0:4 (for MRADlp); and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID N0:2 (i:or HRADlp) or from about 227 to about 257 in SEQ ID N0:4 (for MRADIp).
The invention also relates to methods of producing an isolated HRAD lp-or MRAD lp-specific antibody comprising innmunizing an animal with the above-described isolated BRAD l p or MRAD l p polypeptides, and isolating an HRAD 1 p- or MR.AD 1 p-specific antibody from the animal. The invention is also directed to isolated HRAD 1 p- or MRAD 1 p-specific antibodies produced by these methods. The antibodies of the invention may be polyclonal or monoclonal antibodies, and may be detectably labeled or immobilized on a solid support.
The invention also relates to antisense oligonucleotides which are complementary to an HRADl or a MRADI InRNA sequence corresponding to a portion of the above-described HRADl or MRADl nucleic acid molecules.
Preferred such antisense oligonucleotides may be 15-mers to 40-mers and may be DNA molecules or derivatives thereof such as DNA phosphorothioate molecules. The invention also relates to pharmaceutical compositions comprising one or more of these antisense oligonucleotides and a pharmaceutically acceptable carrier or excipient therefor.
The invention also relates to ribozymes comprising target sequences which are complementary to an HRADI or a MRADI mRNA sequence corresponding to a portion of the above-described HRADI or MRADl nucleic acid molecules. The invention also relates to pharmaceutical compositions comprising these ribozymes and a pharmaceutically acceptable earner or excipient therefor.
The invention is also directed to transgenic animals comprising one or more of the above-described isolated mammalian RADI nucleic acid molecules or mutants, derivatives or variants thereof. Preferred such transgenic animals may be mice comprising an altered HRA.DI or MRADI gene that includes _$_ mutations rendering the gene functionally inactive, particularly wherein the mutations comprise deletion of one or more nucleotides from, or insertion or substitution of one or more nucleotides into, a wildtype HRADI or MRADI gene such as that having the nucleotide sequence set forth in SEQ ID NO: l or SEQ
ID
N0:3, respectively.
The invention is also directed to methods for determining the sensitivity of a first animal (preferably a mammal such as a mouse or human) to a DNA-damaging agent (such as radiation or a chemical), or for diagnosing a disorder in a first animal suffering therefrom or predisposed thereto wherein the disorder is characterized by increased sensitivity of the first animal to a DNA-damaging agent. Preferred such methods of the invention comprise:
(a) obtaining a first biological sample from the first animal and a second biological sample from a second animal of known sensitivity to the DNA-damaging agent;
(b) determining the level of expression of a mammalian RADI gene, such as an HRADI or an MRADl gene, in the first and second biological samples;
and (c) comparing the level of mamnnalian RADl gene expression in the first biological sample to the level of mammalian RADI gene expression in the second biological sample. According to~ the invention, a lower level of mammalian RADI gene expression in the fimt biological sample relative to that of the second biological sample indicates a hdgher level of sensitivity to a DNA-damaging agent in the first animal relative to that of the second animal. In the practice of this aspect of the invention, the determination step (b) may be accomplished by a technique, such as northern blotting, that measures the amount of mammalian RADl mRNA (such as HRAL)1 or MRADl mRNA) present in the first and second biological samples. Alternatively, the determination step (b) may be accomplished by a technique that measures the level or activity of mammalian RADIp polypeptide (such as F(RADlp or MRADlp polypeptide) present in the first and second biological samples; such a technique may be an immunological technique which may comprise contacting the first and second biological samples with one or more antibodies that bind specifically to one or more mammalian RADIp (such as HRADlp or MRADIp) polypeptides present in the first and second biological samples.
The invention is also directed to methods of treating or preventing a disorder, such as a cancer, in a first animal suffering therefrom or predisposed S thereto, comprising:
(a) obtaining a first biological sample from the first animal and a second biological sample from a second animal of known sensitivity to a DNA-damaging agent;
(b) determining the level of mammalian RADI gene expression, such as that of an HRADl gene or an MRADl gene, in the first biological sample relative to that of the second biological sample; and (c) treating the first animal, in which the level of mammalian RADI
gene expression in the first biological sample is different from that of the second biological sample, by a technique that cures, delays or prevents the development of, or induces remission of, the disorder. One suitable technique that cures, delays or prevents the development of, or induces remission of, the disorder, may be selected from the group consisting of (a) chemotherapy, (b) radiation therapy, (c) surgery and (d) a combination of two or more of these techniques in (a), (b) and (c). Alternatively, the technique that cures, delays or prevents the development of, or induces remission of, the disorder may comprise introducing into the first animal a composition comprising one or more of the above-described mammalian RADl (e.g., HRADI or MRADI ) antisense oligonucleotides or one or more of the above-described mammalian RADI (e.g., HRADI orMRADI ) ribozymes, wherein the introduction of the composition into the first animal induces a decrease in the expression of mammalian RAD1 (e.g., HRADI or MRADI ) in the animal. Finally, the technique that cures, delays or prevents the development of, or induces remission of, the disorder may comprise introducing into the first animal a composition comprising one or more of the above-described isolated HRAD1 nucleic acid molecules, wherein the introduction of said composition into the first animal induces an increase or a decrease in the expression of mammalian RADI (e.g., HRADl or MRADI ) in the animal. In preferred such methods of the invention, the composition introduced into the animal further comprises a pharmaceutically acceptable carrier or excipient, or a vector or a virion which may be derived from a retrovirus, an adenovirus or an adeno-associated virus. According to the invention, any of the above methods and compositions may be used in conjunction with one or more of the above-described conventional therapies, such as chemotherapy or radiation therapy. Disorders capable of being treated or prevented by the methods of the invention include cancers, and animals suitably treated by these methods include mammals, preferably humans.
The invention is also directed to methods of identifying and/or isolating mammalian, particularly human and rodent (particularly mouse), cell cycle checkpoint control polypeptides, based on the ability of such polypeptides to interact specifically with HRAD 1 p and/or M:RAD 1 p. Methods according to this aspect of the invention may comprise one or more steps, including, for example:
(a) immobilizing the HR.ADIp or MRADIp on a solid support;
(b) contacting the immobilized FIRAD 1 p or MRAD 1 p with a sample containing one or more cell cycle checkpoint control polypeptides that interact with HR.ADlp and/or MRAD lp, under conditions favoring the interaction of the one or more polypeptides with the immobiliized HRADlp or MRADIp; and (c) releasing the one or more cell cycle checkpoint control polypeptides from the immobilized HRAD 1 p and/or MRAD 1 p, thereby isolating the one or more cell cycle checkpoint control polypeptides.
In an alternative such method, one or more cell cycle checkpoint control polypeptides may be identified or isolated using the anti-HRADlp and/or anti-MRAD 1 p antibodies of the invention, by co-immunoprecipitation of HRAD 1 p or MRAD 1 p complexed with the one or more cell cycle checkpoint control polypeptides, as described in more detail in Examples 10-12 herein.
The invention is also directed 1;o cell cycle checkpoint control polypeptides that are isolated and/or identified according to these methods of the invention.

Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.
BRIEF DESCRIPTION OF' THE DRAWINGS
Figure 1 is a depiction of the complete cDNA nucleotide sequence (SEQ
ID NO:1 ) and deduced complete amino acid (SEQ ID N0:2) sequence of HRADl.
Figure 2 is a depiction of the complete cDNA nucleotide sequence (SEQ
ID N0:3) and deduced complete amino .acid (SEQ ID N0:4) sequence of MRADI. Shown is the complete MRAD~! sequence containing a 224 by S' untranslated region (UTR), an 840 by codling region, and a 328 by 3' UTR, flanked at the 5' and 3' ends by 6 by EcoRI sites.
Figure 3 is a depiction of the ali~~ed amino acid sequences of the deduced HRAD1 polypeptide (HRADIp I;SEQ ID N0:2)) and the MRAD1 polypeptide (MRAD 1 p (SEQ ID N0:4)) with other members of the Rad 1 p family of proteins (U. maydis RECIp (SEQ ID N0:25), S. pombe Radlp (SEQ ID
N0:26), and S. cerevisiae RAD 17p (SEQ ID N0:27), indicating the regions of amino acid sequence identity between the amino acid sequences of HRAD 1 p and MRADlp and other members of the Radlp family. Numbers on the right indicate the numbering of the final amino acid on each line. Identical residues in z 80% of the sequences are highlighted in dark grey. Conserved residues (I/L/V/M, D/E, S/T, A/G, N/Q, R/H/K, and W/F/Y) in z 80% of the sequences are highlighted in light grey. The proposed e~:onuclease and leucine-rich regions (Thelen, M.P., et al., J. Biol. Chem. 269:747-54 ( 1994); Onel, K., et al., Genetics 143:165-74 (1996)) are indicated.
Figure 4 is a depiction of the aligned amino acid sequences of the deduced HRADlp polypeptide (SEQ ID N0:2) and the deduced MRADIp polypeptide (SEQ ID N0:4). Amino acid residues that are conserved between the two polypeptides are designated with an asterisk; those residues where conservative differences between the two po~lypeptides exist are designated with a dot; and those with non-conservative differences are blank.
Figure 5 is a composite of line graphs of the relative viabilities of radl -1 mutants of S. pombe versus radiation dose. Each data point corresponds to the average taken over three plates and error bars indicate the standard deviation of the three values. O, wild type; 0, HRADl sense in radl-1; O, HRADI antisense in radl -1. Error bars cannot be seen for data points where they are smaller than the data markers. Fig. 5A: cells irradiated with UV light; Fig. 5B: cells irradiated with y radiation.
Figure 6 is a composite of line graphs demonstrating the ability of HRADI to rescue the checkpoint defects of radl mutants at varying doses of LTV-irradiation. Fig. 6A: checkpoint-deficient vector-transformed control cells;
Fig. 6B: checkpoint-proficient yeast expressing Radlp; Fig. 6C: checkpoint-proficient yeast expressing HRADlp. O, 0 J/mz; D,10 J/m2; D, 30 J/m2. These data are representative of three independent experiments.
Figure 7 is a line graph of the ability of HRADl expression to restore hydroxyurea (HL~ resistance to radl:: ura4-~ yeast.
Figure 8 is a composite demonstrating the interaction between HRAD1 and hHUS 1. Fig. 8A: ,~'. cerevisiae strain HF7c was transformed with the indicated GAL4 fusion plasmids and plated on media selecting for co-transformants. Single colonies were subcultured onto selective media in the presence (lefthand plate) or absence (righthand plate) of histidine. Growth in the absence of histidine is indicative of a protein::protein interaction, as demonstrated by the p53:SV40 T-Ag positive control (lower left quadrant). When pGBT9-HRAD9 and pGAD-hHUS 1 were cotransformed separately with the corresponding empty vector, no growth on h~iple dropout medium (see Materials and Methods, below) was observed (upper l:wo quadrants). Expression of both HRAD 1 and hHUS 1 GAL4 fusions were required for viability in the absence of histidine (bottom right quadrant). Fig. 8B: COS-1 cells were transiently co-y transfected with constructs expressing FLACi epitope-tagged HRAD 1 (HR.AD 1 fj and myc epitope-tagged hHUS 1 (hHUS 1 "~ (:lane 1 ), hHUS l m and FLAG epitope-tagged hepatic leukemia factor (HLFf) (lane 2), or HRAD 1 f and a myc-tagged n-terminal deletion of the fer oncogene (FerON'm) (lane 3). After harvesting, lysates were immunoprecipitated with anti-myc ("cc-myc") 9E 10 monoclonal antibody (Sigma, and gift of Dr. Peter Greer, Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada) or a- FLAG M2 monoclonal antibody (Sigma). Two aliquots from each sample were subjected to electrophoresis through two identical polyacrylamide gels, one of which was used for an anti-FLAG (a-Flag) western blot and the other for an anti-myc (a-Myc) western blot.
Figure 9 is a composite demonstrating the interaction between HRAD9 and HRAD 1. Fig. 9A: Yeast two-hybrid assay, performed as in Figure 8A, using pGBT9-HRAD9 and pGAD-HRAD 1 GAL4 fusion constructs. Co-expression of HRAD9 and HR.AD 1 fusions failed to assemble a functional GAL4, and hence did not produce viable cells in the absence ~of histidine (Figure 9A, lower right quadrant). Fig. 9B: Co-immunoprecipitation of HRAD1 and HRAD9. COS-1 cells were transiently co-transfected with constructs expressing FLAG epitope-tagged HRAD 1 (HRAD 1 f) and myc epitope-tagged HR.AD9 (HR.AD9m) (lane 1 ), HRAD9m and HLFf (lane 2), or HRAD 1 f and Fer~Nm (lane 3). After harvesting, lysates were immunoprecipitated with a-myc 9E10 monoclonal antibody or a-FLAG M2 monoclonal antibody. Two aliquots from each sample were subj ected to electrophoresis through two identical polyacrylamide gels, one of which was used for an anti-FLAG (a-Flag) western blot and the other for an anti-myc (a-Myc) western blot.
Figure 10 is a composite demonstrating the preferential association of HRAD1 with phosphorylated HRAD9. (:OS-1 cells were transfected with HRAD9my~ expressing construct, and harvf;sted 48 hours later. Alternatively, HeLa cells were simply harvested at 50% confluence. Cells were lysed and immunoprecipitated as in Figure 8 with a-myc or a-HRAD9 antibodies, respectively. Immunoprecipitates were either untreated, or treated with calf intestinal phosphatase (CIP) in the presence or absence of the phsophatase inhibitor sodium orthovanadate (V04). Samples were then subjected to SDS-PAGE, blotted onto nitrocellulose, and immtaroblotted with a-myc or a-HR.AD9 antibodies. Fig. 10A: banding pattern of HltAD9my~ in immunoblots of COS-1 cells, demonstrating multiple phosphorylation states of HRAD9my~. Fig lOB:
banding pattern of HRAD9my~ in HeLa cells, indicating that endogenous HRAD9 is phosphorylated in HeLa cells. Fig. l OC: co-immunoprecipitations of HRAD 1 and HRAD9 in the presence and absence of CIP and/or V04, indicating that the most heavily phosphorylated forms of HltAD9 are preferentially bound by HRAD 1.
DETAILED DESCRIPTION I~F THE INVENTION
Overview The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding mammalian homologues or orthologues of the yeast radl + polypeptide (i.e., "marrunalian RADlp" polypeptides). In particular, the invention is directed to isolated nucleic acid molecules encoding a human RADlp (HRADIp) polypeptide, and to isolated nucleic acid molecules encoding a murine RAD 1 p (MRAD 1 p) pol~,~peptide. The HRAD 1 p polypeptide of the present invention shares about 27% overall amino acid sequence identity and about 53% amino acid sequence similarity with certain members of the Radlp family of yeast cell cycle checkpoint control polypeptides (Figure 3), although the sequence identity and similarity vary within individual domains of the polypeptide as described in detail in the Examples below. The nucleotide sequence of HRADI shown in Figure 1 (SEQ ID NO:1) was obtained by sequencing a cDNA clone prepared from a human transformed keratinocyte cDNA library (HaCaT); the sequence was df;posited with GenBank and assigned GenBank Accession Number AF 011905, and the clone was deposited on at and assigned accession number Although a mammalian homologue of the yeast radl + gene was initially isolated from a human cDNA
library and was designated HRADl , the presE;nt invention also provides a marine homologue ofHRADl , referred to throughout: this specification as "MRADI ,"
that is expressed in marine cells and tissues. The nucleotide sequence of MRADI
shown in Figure 2 (SEQ ID N0:3) was deposited with GenBank and assigned GenBank Accession Number AF 038841, and the clone was deposited on at and assigned accession numbc;r It will therefore be understood by one of ordinary skill in the art that the term " mammalian RADl "
as used herein refers to isolated mammalian RADl nucleic acid molecules or polynucleotides, or mammalian RADIp polypeptides and antibodies, that may originate from any mammal, including those of human or marine origin (i. e., HRADl and HRADlp, or MRADl and MIRADIp, respectively), and that the present invention thus encompasses RADI nucleic acid molecules and polynucleotides, and RAD 1 p polypeptides and antibodies, of mammalian, most particularly of human and marine, origin.
Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using a T7 sequencing kit containing T7 DNA polymerase (Pharnnacia Biotech) according to the instructions of the manufacturer, as described in more detail in the Examples below. It will be understood by one of ordinary skill, however, that the nucleotide sequences of the nucleic acid molecules of the invention could also be determined by manual DNA sequencing such as dideoxy sequencing, according to methods that are routine in the art (Sanger., F., and Coulson, A.R., J.
Mol. Biol.
94:444-448 (1975); Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)), or by automated sequencing such .as by using an Applied Biosystems Automated Sequenator according to the maJlufacturer's instructions. All amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by conceptual translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by these approaches, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by such methods are typically at least about 90% identical, more typically at least about 95% to at least about 99.9%
identical to the actual nucleotide sequence oiF the sequenced DNA molecule. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be .completely different from the amino acid sequence actually encoded by the seqwenced DNA molecule, beginning at the point of such an insertion or deletion.
Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucle;otides (abbreviated A, G , C and T).
However, by "nucleotide sequence" of a nuclleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotidea (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (I~. For instance, reference to HRADI
or MRADl RNA molecules having the sequence of SEQ ID NO:I or SEQ ID
N0:3, respectively, set forth using deoxyribonucleotide abbreviations is intended to indicate RNAs molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO:1 or SEQ II) N0:3 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
Using the information provided herein; such as the nucleotide sequences in Figures l and 2, nucleic acid molecules ~of the present invention encoding a mammalian RADIp polypeptide, such as; HR.ADlp or M>2ADlp, may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. As used herein, an "HRADIp polypeptide" means a polypeptide that is encoded by a polynucleotide comprising the nucleotide sequence shown in Figure 1 (SEQ ID NO:1 ), or that has an amino acid sequence as set forth in Figure 1 (SEQ ID N0:2), or a fragment thereof.
Analogously, an "MRADIp polypeptide" as used herein refers to a polypeptide that is encoded by a polynucleotide comprising the nucleotide sequence set forth in SEQ ID N0:3, or that has an amino acid sequence as set forth in SEQ ID
N0:4, or a fragment thereof. Preferred cloning and screening methods used in the invention include PCR-based cloning methods, such as reverse transcriptase-PCR
(RT-PCR) using primers such as those described in the Examples below.
Illustrative of the invention, the determined nucleotide sequence of the coding segment (849 base pairs) of the HRA1)1 cDNA is shown in Figure 1 (SEQ
ID NO:1 ). The predicted 282 amino acid HItAD lp polypeptide encoded by this coding sequence has an amino acid sequence as set forth in Figure 1 (SEQ ID
N0:2), and a deduced molecular weight of about 32 KDa. Analogously, the determined nucleotide sequence of the codiing segment (843 base pairs) of the MRADl cDNA is shown in Figure 2 (SEQ ID N0:3). The predicted 280 amino acid MRADIp polypeptide encoded by this coding sequence has an amino acid sequence as set forth in Figure 2 (SEQ II) N0:4), and a deduced molecular weight of about 32 KDa Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA,, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
For example, recombinant DNA molecules .contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, and those DNA molecules purific;d (partially or substantially) from a solution whether produced by recombinant DNA or synthetic chemistry techniques. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. However, it is intended that "isolated" as used herein does not include the mammalian RADI cDNA present in a mammalian cDNA library or in a preparation of purified or isolated total genomic DNA containing the mammalian IZ.4D1 gene.
The nucleic acid molecules of the. present invention further include genetic constructs comprising one or more mammalian RADI DNA sequences (such as HRADI or MRADI ) operably linked to regulatory DNA sequences (which may be heterologous regulatory sequences), such as promoters or enhancers as described below. Upon expression of these DNA sequences in host cells, preferably in bacterial, fungal (including yeast), plant or animal (including insect or mammalian) cells, one or more mammalian RAD lp polypeptides, such as HRADIp or MRADIp, may be produced. In such constructs, the regulatory sequences may be operably linked to a mammalian RADI polynucleotide encoding a mature mammalian RADIp polypeptide or any of its variants, precursors, fragments or derivatives described herein. For example, an HRADl variant may include one or more polynucleotides having a nucleic acid sequence that is similar or complementary to substantially all or a portion of a nucleic acid molecule having a nucleic acid sequence as shown in Figure 1 (SEQ ID NO:1 ).
Analogously, an MRADI variant may include one or more polynucleotides having a nucleic acid sequence that is similar or complementary to substantially all or a portion of a nucleic acid molecule having a nucleic acid sequence as shown in Figure 2 (SEQ ID N0:3). As used herein, the term "substantially all"
of a nucleic acid molecule or a polypeptide means a portion of the nucleic acid molecule or polypeptide that contains greatc;r than about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of a reference nucleotide sequence, for example the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID N0:3, or of a reference polypeptide amino acid sequence, for example the polypeptide amino acid sequences shown in SEQ ID N0:2 and SEQ ID N0:4. As used herein, the terms "a portion" or "a fragment" of a nucleic acid molecule or a polypeptide means a segment of a polynucleotide or a polypeptide comprising at least S,10 or 1 S, and more preferably at least 20, contiguous nucleotides or amino acids of a reference polynucleotide one polypeptide (for example, the polynucleotides and polypeptides shown in Figure 1 (SEQ ID NOs: 1, 2) and Figure 2 (SEQ ID NOs: 3, 4)), unless otherwise specifically defined below.
Isolated nucleic acid molecules of the present invention include (a) DNA
molecules encoding a mammalian RAD lp p~olypeptide, for example the HRADI
S DNA molecule having a nucleotide sequence corresponding to that depicted in Figure 1 (SEQ ID NO:1 ) and the MRADl :DNA molecule having a nucleotide sequence corresponding to that depicted in Figure 2 (SEQ ID NO:3); (b) DNA
molecules comprising the coding sequence fir a mammalian RAD lp polypeptide, for example the HRADIp polypeptide shown in Figure 1 (SEQ ID N0:2) and the MRADlp polypeptide shown in Figure 2 (SEQ ID N0:4); and (c) DNA
molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy ofthe genetic code, still encode a mammalian RAD lp polypeptide. Since the genetic code is well known in the art, it is routine for one of ordinary skill in the art to produce the degenerate variants described above without undue experimentation.
In another aspect, the invention provides an isolated HRADl nucleic acid molecule having a nucleotide sequence as set forth in Figure 1 (SEQ ID NO:1 ), an isolated MRADl nucleic acid molecule leaving a nucleotide sequence as set forth in Figure 2 (SEQ ID N0:3), or a nucleic acid molecule having a sequence similar or complementary to substantially all or a portion of such nucleic acid molecules. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the mammalian RAL)1 (e.g., HRADl or MRADI ) gene in animal (especially mammalian, including human and murine) tissues and cells, for instance by northern blot analysis.
Nucleic acid molecules of the present invention which encode a mammalian RADIp polypeptide may include, but are not limited to, those encoding the amino acid sequence of the mal:ure polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding the about 20-amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example introns and non-coding 5' and 3' sequences, such as the transcribed, untranslated regions (UTRs) or other 5' flanking sequences that nnay play a role in transcription (e.g., via providing ribosome- or transcription factor-binding sites), mIRNA
processing (e.g. splicing and polyadenylation signals) and stability of mRNA; the coding sequence for the mature mammalian RADIIp polypeptide operably linked to a regulatory DNA sequence, particularly a hetcrologous regulatory DNA sequence such as a promoter or enhancer; and the; coding sequence for the mature mammalian RADIp polypeptide linked to one or more coding sequences which code for amino acids that provide additional functionalities. Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain embodiments of this aspect of the invention, the marker amino acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE
vector (Qiagen, Inc.), among others, many of which are commercially available.
As described for instance in Gentz et al., Prnc. Natl. Acad. Sci. USA 86:821-( 1989), hexa-histidine provides for convenient purification of the fusion protein.
The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hema;gglutinin protein, which has been described by Wilson et al., Cell 37: 767 ( 1984). Yet another useful marker peptide for facilitating the purification of a mammalian RAD 1 polypeptide is glutathione S-transferase (GST) encoded b;r the pGEX fusion vector (see, e.g., Winnacker, From Genes to Clones, New Fork: VCH Publishers, pp. 451-481 ( 1987)). As discussed below, other such fusion proteins include the mammalian RAD1 polypeptide fused to immunoglobulin Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the mammalian IRADIp polypeptides of'the invention. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (see Lewin, B., Ed., Genes 11, John Wiley & Sons, New York (1985)). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or insertions. The substitutions, delletions or insertions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are substitutions, additions and deletions, which alter the properties and activities of the mammalian RAD lp polypeptide protein or portions thereof; for example, most particul2~rly preferred in this regard are those substitutions, deletions and insertions vrhereby the mammalian RAD 1 p polypeptide is rendered substantially reduced in activity. As used herein, the term "substantially reduced in activity" means the mammalian RADlp polypeptide or mutant mammalian RAD 1 p polypeptide demonstrates an activity level not greater than about SO%, 40%, 30%, 20%,10%, 5%,1 %, 0.1 % or 0.01 %, of the level demonstrated by a wildtype mammalian RAD 1 polypeptide such as the wildtypes of HRAD 1 or MR.AD 1. In practice, whether a mammalian RAD lp polypeptide or a mutant mammalian RAD 1 polypeptide demonstrates an activity level not greater than about SO%, 40%, 30%, 20%,10%, S%,1 %, 0.1 % or 0.01 %, of the level demonstrated by a wildtype malrunalian RAD 1 p polypeptide may be determined by any number of assays measuring the sensitivity of mammalian cells, expressing the RAD 1 p or mutant RAI~ 1 p polypeptides, to DNA damage.
Such assays will be familiar to one of ordinary skill; analogous assays in yeast cells are described in detail in the Example,. below.
Further embodiments of the invention include isolated HRADl nucleic acid molecules comprising a polynucleotide :having a nucleotide sequence at least about 65% identical, and more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%
identical, to:
(a) the nucleotide sequence set forth in SEQ ID NO:1;

(b) a nucleotide sequence encoding the HRAD lp polypeptide having the complete amino acid sequence set forth in SEQ ID N0:2;
(c) a nucleotide sequence encoding the IiRADIp polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on at and has accession number ;
(d) a nucleotide sequence of a po lynucleotide which hybridizes under stringent conditions to a polynucleotide haviing the nucleotide sequence set forth in SEQ ID NO:1; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d), or a fragment thereof.
Additional embodiments ofthe invention include isolatedMRADl nucleic acid molecules, comprising a polynucleoti~de having a nucleotide sequence at least about 65% (more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) identical to a reference sequence selected from the group consisting of (a) the nucleotide sequence set forth in SEQ ID N0:3;
(b) a nucleotide sequence encoding the MRAD lp polypeptide having the complete amino acid sequence set forth in SEQ ID N0:4;
(c) a nucleotide sequence encoding the MRAD 1 polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on at and has accession number ;
(d) a nucleotide sequence of a po lynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence set forth in SEQ ID N0:3; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d), or a fragment thereof.

By "stringent hybridization conditions" as used herein is meant overnight incubation at 42 °C in a solution comprising: :50% formamide, Sx SSC
(1X SSC =
150 mM NaCI, lSmM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate;, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 °C.
By a polynucleotide having a nucleotide sequence at least, for example, 65% "identical" to a reference nucleotide sequence encoding a mammalian RADIp polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to 35 point mutations per each 100 nucleotides of the reference nucleotide se-quence encoding the mammalian RADIp polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 65% identical to a reference nucleotide sequence, up to 35% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, ar a number of nucleotides up to 35% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for instance, the HRADI nucleotide sequence shown in Figure 1 (SEQ ID NO:1) or the MRADI nucleotide sequence shown in Figure 2 (SEQ ID
N0:3), can be determined conventionally using known computer programs such as BLAST (Washington, DC) or BEST:EIT (Genetics Computer Group, University Research Park, Madison, WI). Wlnen using BLAST, BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 65% identical to a reference sequence according to the present invention, the parameters are set such that the; percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 35% of the total number of nucleotides in the reference sequence are allowed.
The present invention is directed to nucleic acid molecules at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96°/~, 97%, 98% or 99% identical to the HRADI nucleic acid sequence shown in Fig~:~re 1 (SEQ ID NO:1 ) or the MRADl nucleic acid sequence set forth in SEQ :ID N0:3, and fragments thereof, irrespective of whether they encode a polypeptide having RAD 1 p activity.
This is because even where a particular nucleic acid molecule does not encode a polypeptide having RADIp activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a p~olypeptide having RADIp activity include, inter alia, (1 ) isolating the mammalian RADl gene (e.g., the HRADI
or MRADI gene) or allelic variants thereof in a genomic DNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the mammalian RADl gene, as described for human gene localization in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New ~.'ork ( 1988); and (3) northern blot analysis for detecting RAD 1 mRNA expression in specific mammalian tissues.
Of course, due to the degeneracy of flue genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the HRADI nucleic acid sequence shown in Figure 1 (SEQ ID NO:1) or the MiQADl nucleic acid sequence shown in Figure 2 (SEQ ID N0:3), and fragments thereof, will encode a polypeptide having mammalian RAD 1 p polypeptide structure and/or activity. In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled wtisan even without performing the above described comparison assay. It will be further recognized by one of ordinary skill in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having RADlp polypeptide structure and/or activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or unlikely to significantly affect protein function (e.g., rep:lacing one aliphatic amino acid with a second aliphatic amino acid). For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U., et al., Science 247:1306-1310 (1990), and the references cited therein.
As noted above, the invention also provides fragments of the above-described nucleic acid molecules. Preferred nucleic acid fragments of the present invention include isolated nucleic acid molecules encoding epitope-bearing portions of the mammalian RADIp polypeptides of the invention, such as HRADlp and MRADlp. In particular, such HRADl nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in Figure 1 (SEQ ID NC1:2); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about 97 in Figure 1 (SEQ ID N0:2); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in Figure 1 (SEQ ID N0:2); a polypeptide having an amino acid sequence consisting essentially of amino acid residuf;s from about 159 to about 177 in Figure 1 (SEQ ID N0:2); and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in Figure 1 (SEQ ID N0:2). Analogously, such MRADI nucleic acid fragments may include nucleic acid molecules encoding: a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in Figure 2 (SEQ ID N0:4); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about in Figure 2 (SEQ ID N0:4); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in Figure 2 (SEQ ID N0:4); a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in Figure 2 (SEQ ID N0:4); and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in Figure 2 (SEQ ID N0:4). The inventors contemplate that the above polypeptide fragments are antigenic regions of the predicted HRAD 1 p and MRAD 1 p polypeptides. Methods for determining other such epitope-bearing portions of the mammalian RAD1 polypeptides are described in detail below.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridlizes under stringent hybridization conditions to substantially all or a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, an HRADI nucleic acid molecule having a nucleotide sequence as set forth in Figure 1 (SEQ ID NO:1 ) or an MRADl nucleic acid molecule having a nucleotide sequence as set forth in Figure 2 (SEQ ID N0:3).
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 30 nucleotides, and more preferably at least about 40 nucleotides, still more preferably at least about 50 nucleotides, and even more preferably about 50-90 nucleotides of the reference polynucleotide. These hybridizing polynucleotides are useful as diagnostic probes and primers as discussed above and in more detail below.
Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., a nucleic acid molecule consisting of the HRADI coding sequence, having the nucleotide sequence se;t forth in Figure 1 (SEQ ID NO:1 ), or a nucleic acid molecule consisting of the ~TRADI coding sequence, having the nucleotide sequence set forth in Figure 2 (SEQ ID N0:3)), for instance, a portion about 100-800 nucleotides in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as axe polynucleotides corresponding to most, i f not all, of the nucleotide sequence as shown in Figure 1 (SEQ ID NO:1) or Figure 2 (SEQ ID N0:3). By a portion of a polynucleotide of "at least 30 nucleotides in length," for example, is intended or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the HRADl nucleotide sequence as shown in Figure 1 (SEQ
30 ID NO:1) or the MRADl nucleotide sequence as shown in Figure 2 (SEQ ID
N0:3)). As indicated, such portions are usf;ful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd Ed., Sambrook, J., et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), the entire disclosure of'which is hereby incorporated herein S by reference.
Since a determined nucleotide sequence encoding an HR.ADlp polypeptide is provided in Figure 1 (SEQ ID NO:1 ) and a determined nucleotide sequence encoding an MRADIp polypeptidie is provided in Figure 2 (SEQ ID
N0:3), generating polynucleotides which hybridize to a portion of a mammalian RADl cDNA molecule, particularly to a portion of an HRADl cDNA molecule or an MRADl cDNA molecule, would be routine to the skilled artisan. For example, restriction endonuclease cleavage; or shearing by sonication of the mammalian RADI cDNA clone could easily be used to generate DNA portions of various sizes which are polynucleotides that hybridize to a portion of the mammalian RADl cDNA molecule. Alternatively, the hybridizing polynucleotides of the present invention could be generated synthetically according to known techniques. Thus, while the present invention specifically relates to RADl nucleic acid molecules and polypeptides from human and mouse, one of ordinary skill could easily generate and/or isolate homologues or orthologues of the HRADl and MRADl nucleic acid molecules and polypeptides of the invention from other organisms (parl:icularly other mammals) using the HRADl nucleotide sequence (SEQ ID NO:1 ) and HRAD 1 p amino acid sequence (SEQ ID N0:2), and the MRADI nucleotide sequence (SEQ ID N0:3) and MRADIp amino acid sequence (SEQ ID N0:4), described herein and routine molecular biology methods, e.g., screening; of cDNA libraries, that are well-known in the art and described in standard protocols (see, e.g., Molecular Cloning, A Laboratory Manual, 2nd Ed., Sambrook, J., et al., eds., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989)). Mammalian tissue cDNA and genomic libraries that may be useful in isolating mammalian RADI nucleic acid molecules are commercially available, for example from Clontech (Palo Alto, California).

Vectors and Host Cells The present invention also relates to genetic constructs comprising the isolated nucleic acid molecules of the invention, or fragments thereof, operably linked to regulatory DNA sequences as described in detail below, vectors which comprise these genetic constructs or the isolated DNA molecules of the present invention, and host cells which comprise these vectors. In addition, the invention relates to the production of mammalian RAI) 1 p polypeptides (such as HRAD 1 p or MRAD 1 p polypeptides) or fragments therf;of by recombinant techniques using these vectors and host cells.
Vectors comprising the genetic constructs or the isolated DNA molecules or fragments of the present invention may be introduced into host cells using well-known techniques such as infecaion, transduction, transfection, electroporation and transformation. The vector may be, for example, a phage, plasmid, viral or retroviral vector, and is preferably an expression vector as described below. Retroviral vectors may be replication-competent or -defective.
In the latter case, viral propagation generall;r will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced into mammalian or avian cells in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid (e.g., LIPOFECTAMINETM; Life Technologies, Inc.; Rockville, Maryland) or in a complex with a virus (such as an adenovirus; see U.S. Patent Nos. 5,547,932 and 5,521,291) or components of a virus (such as viral capsid peptides). If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then used to infect host cells.
Preferred are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate traps-acting factors may be supplied by the host, by a complementing vector or by thE; vector itself upon introduction into the host.
In certain preferred embodiments in this regard, the vectors provide for specific expression, which may be ind.ucible and/or cell type-specific.
Particularly preferred among such expressiion vectors are those inducible by environmental factors that are easy to mmipulate, such as temperature and nutrient additives.
Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial S plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
In one embodiment, an isolated nucleic acid molecule of the invention or fragment thereof may be operably linked to am appropriate regulatory sequence, preferably a promoter such as the phage lambda PL promoter, promoters from T3, T7 and SP6 phages, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LT'Rs and derivatives thereof, to name a few. Other suitable promoters will be ls~own to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiation codon (AUG) at the beginning and a termination codon (LTAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated above, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase (dhfr) or neomycin (neo) resistance for eukaryotic cell culture and tetracycline (tet) or ampicillin (amp) resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate ho sts include, but are not limited to, bacterial cells, such as Escherichia spp. cells: (particularly E. coli), Bacillus spp.
cells (particularly B. cereus, B. subtilis and B. megaterium), Streptomyces spp.
cells, Salmonella spp. cells (particularly S. typhimurium) andXanthomonas spp.
cells; fungal cells, including yeast cells such as Saccharomyces spp. cells;
insect cells such as Drosophila S2, Spodoptera Sf~ or Sf21 cells and Trichoplusa High-Five cells; other animal cells (particularly mammalian cells and most particularly human cells) such as CHO, COS,1VIII-3T3, VERO, HeLa, HeCaT, embryonic stem (ES) cells, Bowes melanoma cells and >:IepG2 and other liver cell lines;
and higher plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHBA, pNHl6a, pNHl8A and pl'JI-I46A, available from Stratagene;
pcDNA3 available from Invitrogen; and pGE:K, pTrxfus, pTrc99a, pET-S, pET-9, pKK223-3, pKK233-3, pDR540 and pRITS available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl, pBK and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Among known bacterial promoters suitable for use in the present invention include the E. coli lacI and lacZ promoters, the T3, T7 and SP6 phage promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RS V), and metallothionein promoters, such as the mouse metallothionein-I
promoter.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAF-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, nucleic acid-coated microproj ectile bombardment or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
In some embodiments, the isolated polynucleotides of the present invention may be operably linked to a regulatory genetic sequence, which may be a homologous or a heterologous regulatory genetic sequence (such as an enhancer, promoter or repressor), to form a genetic construct. Genetic constructs according to this aspect of the invention are intended to encompass not only those comprising a polynucleotide encoding a mature mammalian RAD 1 p polypeptide (such as an HRADpI or an MRADIp p~olypeptide) operably linked to a regulatory DNA sequence, but also those constructs comprising one or more regulatory sequences operably linked to a mammalian RADl polynucleotide fragment which does not encode a mammalian RADlp polypeptide, but which contains a sufficient portion of the mammalian RADI nucleotide sequence (a "targeting fragment") to target the genetic construct to the native mammalian RADl locus upon introduction into a host cell wherein the mammalian RADl gene may be inactive due to repression or nnutation. These constructs may be inserted into a vector as above, and the vectors introduced into a host cell, the genome of which comprises the target gene;, by any of the methods described above. The mammalian RADl polynucleotiide will then integrate into the host cell genome by homologous recombination. :(n the case of a construct comprising a homologous or heterologous regulatory sequence linked to a targeting mammalian RADI polynucleotide fragment, the regulatory sequence will be targeted to the native mammalian RADI lochs in the host cell, and will amplify or de-repress (if the regulatory sequence comprises, for example, a promoter or enhancer) or will inhibit or repress (if the regulatory sequence comprises, for example, a repressor or otherwise integrates into the native regulatory sequence to inhibit or repress (i.e., "knock out")) the expression of the native mammalian RAD1 gene in the host cell, thereby increasing or decreasing the level of mammalian RADI gene expression. Alternatively, such gene targeting may be carried out using genetic constructs comprising the above-described mammalian RADl targeting fragment in the absence of a regulatory sequence; such an approach may be used, for example, to correct or introduce point mutations in the mammalian RADI gene, for example for the purposes of enhancing or inhibiting the activity of the RADI gene in the targeted mammalian cell (see Steeg, C.M., et al., Proc. Natl. Acad. Sci. USA 87(12):46'80-4684 (1990) for a description of the use of such approaches to correcting point mutations in other mammalian genes). Such methods of producing genetic constructs, introducing genes of interest into a host cell via homologous recombination and producing the encoded polypeptides are generally described in U.S. fatentNo. 5,578,461; WO 94/12650 (U.S. Application No. 07/985,586); WO 93/09222 (U.S. Application No.

07/911,535); and WO 90/14092 (U.S. Application No. 07/353,909), the disclosures of which are expressly incorporated herein by reference in their entireties.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act to increase b~anscriptional activity of a promoter in a given host cell-type. Examples of enh;ancers include the SV40 enhancer, which is located on the late side of the repliication origin at by 100 to 270, the cytomegalovirus early promoter enhancer, th.e polyoma enhancer on the late side of the replication origin, and adenovirus enha~ncers. In an alternative embodiment of the invention, transcriptional activation of the mammalian RA.DI gene may be enhanced by inserting one or more concat~unerized elements firom the native human or mammalian RADl promoter into l:he vector.
For secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmaic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be; endogenous to the polypeptide or they may be heterologous signals.
Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, mammalian RADIp polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
The mammalian RADIp polypeptide, such as an HRADlp or an MRADIp polypeptide, may be expressed in a modified form, such as a fusion protein, and may include not only secrE;tion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be; added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate: purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among other purposes, is a familiar and routine technique in the art. A preferred fusion protean comprises a heterologous region from an immunoglobulin that is useful to solubilize proteins. For example, EP 0 464 533 discloses fusion proteins comprising various portions of constant (Fc) region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc portion of a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP 0 232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when the Fc portion proves to be a hindrance to use in therapy, diagnosis or further manufacturing, for example when the fusion protein is to be used as an antigen for immunizations for the preparation of antibodies.
The mammalian ItAD 1 p polype:pt:ide, such as an HRAD 1 p or an MRADIp polypeptide, may be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, lectin chromatography, gel filtration, hydrophobic interaction chromatography, affinity chromatography and hydroxylapatite chromatography. Most preferably, high performance liquid chromatography ("HPL(:") is employed for purification.
Mammalian 12AD1 Polypeptides and Fragments The invention further provides isolatc;d mammalian 1RAD 1 polypeptides.
Polypeptides of the present invention include purified or isolated natural products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, insect, mammalian, avian and higher plant cells.
Examples of mammalian ItAD 1 p polypeptides provided by the invention include, but are not limited to, isolated HRADlp polypeptides and isolated MRADIp polypeptides. Isolated HRADlp polypeptides of the invention include those having the amino acid sequence encoded by a polynucleotide having the nucleotide sequence set forth in Figure 1 (SEQ ID NO:1 ), the complete amino acid sequence in Figure 1 {SEQ ID N0:2), the complete amino acid sequence encoded by a polynucleoti.de contained in the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on at and has accession number , the amino acid sequence encoded by a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence as set forth in Figure 1 (SEQ ID
NO:1), or a peptide or polypeptide comprising a portion or a fragment of the above polypeptides. Analogously, isolated MRAD lp polypeptides of the invention include those having the amino acid sequence encoded by a polynucleotide having the nucleotide sequence set forth in Figure 2 (SEQ ID
N0:4), the complete amino acid sequence in Figure 2 (SEQ ID N0:4), the complete amino acid sequence encoded by a polynucleotide contained in the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on at and has accession number , the amino acid sequence encoded by a polynucleotide: which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence as set forth in Figure 2 (SEQ ID N0:3), or a peptide or polypeptide comprising a portion or a fragment of the above polypepti.des.
As used herein, the terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by (a) peptidyl linkage(s). The term "polypeptide" is used herein to denote chains comprising nine or more amino acid residues, unless otherwise defined in the specific contexts below. As is commonly recognized in the art, all oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus.
It will be recognized by those of ordinary skill in the art that some amino acid sequences of the mammalian RADlp p~olypeptides of the invention can be varied without significant effect on the structure or function of the polypeptides.

If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine structure and activity.
In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the polypeptide.
Thus, the invention further includes variants of the present mammalian RADIp polypeptides, including allelic variants, which show substantial mammalian RAD 1 p polypeptide structural homology or activity, or which include regions of the mammalian RADIp polypeptides such as the portions discussed below. Such mutants may include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule).
Small changes or such "neutral" or "conservative" amino acid substitutions will generally have little effect on activity.
Typical conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxylated residues Ser and Thr; exchange. of the acidic residues Asp and Glu;
substitution between the arnidated residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements amaong the aromatic residues Phe and Tyr.
Thus, the fragment, derivative or ~uialog of the mammalian RADIp polypeptide of the invention, such as the HRADlp polypeptide depicted in Figure 1 (SEQ ID N0:2) ar that encoded by a polynucleotide having a nucleic acid sequence as set forth in Figure 1 (SEQ ID NO:1 ), or such as the MRAD 1 p polypeptide depicted in Figure 2 (SEQ ID N0:3) or that encoded by a polynucleotide having a nucleic acid sequence as set forth in Figure 2 (SEQ ID
N0:3) may be (i) one in which one or more of the amino acid residues are substituted with a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and such substituted amino acid residue may be encoded by the genetic code or may be an amino acid (e.g., desmosine, citrulline, ornithine, etc.) that is not encoded by the genetic code; (ii) one in which one or more of the amino acid residues includes a substituent group (e.g., a phosphate, hydroxyl, sulfate or other group) in addition to the normal "R"
group of the amino acid; (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half life of the polypeptide (for example, polyethylene glycol), or (iv) one in which additional amino acids are fused to the mature polypeptide, such as an immunoglobulin Fc region peptide, a leader or secretory sequence:, a sequence which is employed for purification of the mature polypeptide (such as GST) or a proprotein sequence.
Such fragments, derivatives and analogs are intended to be encompassed by the present invention, and are within the scope of those skilled in the art from the teachings herein and the state of the art at the time of invention.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. Recombinantly produced versions of the mammalian RAD 1 p polypeptides of the invention can be substantially purified by the one-step metriod described in Smith and Johnson, Gene 67: 31-40 (1988). As used herein, the l:erm "substantially purified"
means a preparation of an individual mammalian RADIp polypeptide wherein at least 50%, preferably at least 70%, and more preferably at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (by mass) of contaminating proteins (i. e. , those that are not the individual mammalian R.AD 1 p polypeptide) have been removed from the preparation.
The polypeptides of the present invention include those which are at least about 65% identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%
identical, to the above-described polypeptide;s. For example, preferred HRAD 1 p polypeptides of the invention include those l;hat are at least about 65%
identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at lea~;t about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, to the polypeptide encoded by a polynucleotide h~~ving a nucleic acid sequence as set forth in Figure 1 (SEQ ID NO: l ), to the pol;,rpeptide having the complete amino acid sequence shown in Figure 1 (SEQ ID N0:2), to a polypeptide encoded by a polynucleotide contained in the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on at and has accession number , or to a polypeptide encoded by a polynucleotide hybridizing under stringent conditions to a polynucleotide having the nucleotide sequence set forth in Figure 1 (SEQ ID NO:1 ). Analogously, preferred MRAD 1 p polypeptides of the invention include those that are at least about 65%
identical, more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, to the polypeptide encoded by a polynucleotide having a nucleic acid sequence as set forth in Figure 2 (SEQ ID N0:3), to the polypeptide having the complete amino acid sequence shown in Figure 2 (SEQ ID T10:4), to a polypeptide encoded by a polynucleotide contained in the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on at and has accession number , or to a polypeptide encoded by a polynucleotide hybridizing under stringent conditions to a polynucleotide having the nucleotide sequence set forth in Figure 2 (SEQ ID N0:4). The present polypeptides also include portions or fragments of the above-described polypeptides with at least 30 amino acids and more preferably at least SO amino acids.
By a polypeptide having an amino acid sequence at least, for example, 65% "identical" to a reference amino acidl sequence of a given mammalian RADIp polypeptide is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except. that the polypeptide sequence may include up to 3 S amino acid alterations per e~ich 100 amino acids of the reference amino acid sequence of the mammalian RAIDlp polypeptide. In other words, to obtain a polypeptide having an amino acid ;sequence at least 65% identical to a reference amino acid sequence, up to 35%. of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 3 S % of the total amino acid residues in the reference sequence may be inserted into the reference: sequence. These alterations of the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether a ,given amino acid sequence is, for example, at least 65% identical to the amino acid sequence of a mammalian RADIp polypeptide of the invention can bE; determined conventionally using known computer programs such as those described above for nucleic acid sequence identity determinations, or more yreferably using the CLUSTAL W
program (Thompson, J.D., et al.) Nucleic A~:ids Res. 22:4673-4680 (1994)) as described in detail below in the Examples.
The polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. In addition, as described in detail below, the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies which are useful in diagnostic assays for detecting mammalian RADlp protein expression, as antagonists capable of inhibiting mammalian RAD 1 p protein function or for the isolation of a mammalian RAD 1 p polypeptide.
In another aspect, the present invention provides a peptide or polypeptide comprising an epitope-bearing portion of a ~polypeptide of the invention, which may be used to raise antibodies, particularly monoclonal antibodies, that bind specifically to a mammalian RADIp polypeptide of the invention. The epitope of this polypeptide portion is an immun.ogenic or antigenic epitope of a polypeptide of the invention. An "immuno~;enic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "mtigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes (see, e.g., Geysen et al., Proc. Nztl. Acad. Sci. USA 81:3998- 4002 (1983)).

As to the selection of peptides or polypeptides bearing an antigenic epitope (i. e., that contain a region of a protein molecule to which an antibody can bind), it is well-known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinelly capable of eliciting an antiserum S that reacts with the partially mimicked protE;in (see, e.g., Sutcliffe, J.G., et al., Science 219: 660-666 (1983)). Peptides capalble of eliciting protein-reactive sera are frequently represented in the primar~r sequence of a protein, can be characterized by a set of simple chemical rules, and are not confined to the immunodominant regions of intact proteins (i. e. , immunogenic epitopes) or to the amino or carboxy termini. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer peptides, especially those containing proline residues, usually are effective (Sutcliffe, ,T.G., et al., Science 219:660-666 (1983)).
Epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); sequences containing proline residues are particularly preferred.
Non-limiting examples of epitope-bearing polypeptides or peptides that can be used to generate mammalian RADIp-specific antibodies include certain epitope-bearing regions of the HRADlp polypeptide depicted in Figure 1 (SEQ
ID N0:2) and the MRADIp polypeptide depicted in Figure 2 (SEQ ID N0:4).

Preferred such epitope-bearing regions of HP;ADlp include without limitation a polypeptide having an amino acid sequence c;onsisting essentially of amino acid residues from about 77 to about 86 in Figure 1 (SEQ ID N0:2), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about 97 in Figure 1 (SEQ ID NO:2), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in Figure 1 (SEQ ID N0:2), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in Figure 1 (SEQ ID N0:2), and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in Figure 1 (SEQ ID N0:2). Analogously, preferred such epitope-bearing regions of MRADIp include without limitation a polypeptide having an amino acid sequence consisting essentially of amino acid residues fiom about 77 to about 86 in Figure 2 (SEQ ID N0:4), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about in Figure 2 (SEQ ID N0:4), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in Figure 2 (SEQ ID N0:4), a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in Figure 2 (SEQ ID N0:4), and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in Figure 2 (SEQ ID N0:4). Other epitope-bearing polypeptides or peptides that may be used to generate mammalian RADlp-specific antibodies will be apparent to one of ordinary skill in the art based on the primary amino acid sequence of the HRADIp and MRADIp polypeptides set forth in SEQ ID N0:2 and SEQ ID
N0:4, respectively, via the construction of Kyte-Doolittle hydrophilicity and Jameson-Wolf antigenic index plots of the HRADIp and/or MRADIp polypeptides using, for example, PROTEAN computer software (DNASTAR, Inc.; Madison, Wisconsin).
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies.
Epitope-bearing peptides also may be synl:hesized using known methods of chemical synthesis (see, e.g., U.S. Patent No. 4,631,211; Houghten, R. A., Proc.
Natl. Acad. Sci. USA 82:5131-5135 (1985)).
As one of skill in the art will appreciate, mammalian RAD 1 p polypeptides of the present invention and epitope-bearing fragments thereof may be immobilized onto a solid support, by techniques that are well-known and routine in the art. By "solid support" is intended any solid support to which a peptide can be immobilized. Such solid supports include, but are not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Seph~~rose, agar, starch, nylon, beads and microtitre plates. Linkage of the peptide of the invention to a solid support can be accomplished by attaching one or both ends of the peptide to the support.
Attachment may also be made at one or more internal sites in the peptide.
Multiple attachments (both internal and at tile ends of the peptide) may also be used according to the invention. Attachment can be via an amino acid linkage group such as a primary amino group, a carboxyl group, or a sulfhydryl (SH) group or by chemical linkage groups such as with cyanogen bromide (CNBr) linkage through a spacer. For non-covalent .attachments to the support, addition of an affinity tag sequence to the peptide carp be used such as GST (Smith, D.B., and Johnson, K.S., Gene 67:31 (1988)), polyhistidines (Hochuli, E., et al., J.
Chromatog. 411:77 (1987)), or biotin. Such affinity tags may be used for the reversible attachment of the peptide to the support. Such immobilized polypeptides or fragments may be useful, for example, in isolating antibodies directed against a mammalian RADIp polypeptide, such as an HRADlp or an MRADIp polypeptide, as described below.
As one of skill in the art will also appreciate, mammalian RADIp polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (Ig), resulting in chimeric or fusion polypeptides. These fusion polypeptides facilitate purification and show an increased half life in vivo (EP 0 394 827; Traunecker et al., Nature 331:84- 86 (1988)).
Mammalian RAD1 Antibodies Epitope-bearing peptides and mammalian RADlp polypeptides of the invention may be used to produce antibodies directed against one or more mammalian RADlp polypeptides, such as HRADIp and/or MItADlp polypeptides, according to methods well-laiown in the art. See, for instance, Sutcliffe, J.G., et al., Science 219:660-666 (1983); Wilson et al., Cell 37:

(1984); and Bittle, F.J., et al., J. Gen. Virol, 66:2347-2354 (1985).
Antibodies specific for a mammalian IRADlp polypeptide can be raised against the intact mammalian 1RAD 1 p polypeptide or one or more antigenic polypeptide fragments thereof, such as the epitope-bearing fragments of mammalian IRADIp polypeptides described above. These polypeptides or fragments may be presented together with a carrier protein (e.g. , albumin) to an animal system (such as rabbit or mouse) or, if they are long enough (at least about 25 amino acids), without a Garner.
As used herein, the term "antibody" (Ab) may be used interchangeably with the terms "polyclonal antibody" or "monoclonal antibody" (mAb), except in specific contexts as described below. These terms, as used herein, are meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to a mammalian IZADIp polypeptide (such as an HRADlp or an MIZADlp polypeptide) or a portion thereof. Fab and F( ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl.
Med.
24:316-325 (1983)).
The antibodies of the present invention may be polyclonal or monoclonal, and may be prepared by any of a variety of methods. For example, polyclonal antibodies may be made by immunizing a~n animal with one or more of the mammalian ltADlp polypeptides or portions thereof (including one or more epitope-bearing fragments) of the invention according to standard techniques (see, e.g., Harlow, E., and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (1988); Kaufman, P.B., et al., In: Handbook of Molecular ana! Cellular Methods in Biology and Medicine, Boca Raton, Florida: CRC Press, pp. 468-469 (1995)). In the most preferred method, the antibodies of the yresent invention are monoclonal antibodies (or mammalian RAD 1 p polypeptide-binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology that is well-known in the art (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.
Immunol. 6:511 (1976); Kbhler et al., .Eur. J. Immunol. 6:292 (1976);
Hammerling et al., In: Monoclonal Antibodies and T Cell Hybridomas, New York: Elsevier, pp. 563-681 (1981); Kaufnnan, P.B., et al., In: Handbook of Molecular and Cellular Methods in Biology ~xnd Medicine, Boca Raton, Florida:
CRC Press, pp. 444-467 (1995)).
Alternatively, antibodies capable of binding to one or more mammalian RADIp polypeptides or fragments thereof' may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, mammalian RADlp polyheptide-specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide-specific antibody can be blocked by the mammalian RADIp polypeptide antigen. Such antibodies comprise anti-idiotypic antibodies to the mammalian RAD 1 p polypeptide-specific antibody and can be used to immunize an animal to induce formation of further mammalian RAD 1 p polypeptide-specific antibodies.
In another preferred embodiment of the invention, the present antibodies may be prepared as chimeric antibodies. According to the invention, such chimeric antibodies may comprise an antigen-binding domain (i.e., the region of the antibody binding to a mammalian RADI.p polypeptide, such as an HR.ADlp or an MRADIp polypeptide) from a first species and one or more constant regions from a second species. See U.S. Patent No. 4,816,567, which is directed to methods for the preparation of chimeric antibodies, the disclosure of which is incorporated herein by reference in its entirety.
It will be appreciated that Fab, FI ab')z and other fragments of the antibodies of the present invention may b~e used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Alternatively, mamm~~lian RADlp polypeptide-binding fragments can be produced through the application of recombinant DNA
technology or through synthetic chemistry.
The antibodies of the present invention may be detectably labeled, most preferably with an enzyme, radioisotopic, non-radioactive isotopic, fluorescent, toxin, chemiluminescent or nuclear magnetiic resonance (NMR) contrast agent label. Suitable examples of each of these types of labels are well-known to one of ordinary skill in the art. Typical techniques for binding a label to an antibody are provided, for example, by Kennedy et al'. , Cl in. Ch im. Acta 70:1-31 ( 1976), and Schuss et al., Clin. Chim. Acta 81:1-40 (1977), all of which methods are incorporated by reference herein.
In an additional preferred embodim~snt of the invention, the antibodies produced as described above may be covalently or non-covalently immobilized onto a solid support. By "solid support" is intended any solid support to which an antibody can be immobilized, including but not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, staxch, nylon, beads (including glass, latex, magnetic (including paramagnetic and superparamagnetic) beads) and microtitre plates. Linkage of the antibodies o~f the invention to a solid support can be accomplished by attaching one or more ends of the antibody to the support.
Attachment may also be made at one or snore internal sites in the antibody.
Multiple attachments (both internal and at the ends of the antibody) may also be used according to the invention. Attachment can be via an amino acid linkage group such as a primary amino group, a c~crboxyl group, or a sulfhydryl (SH) group, by chemical linkage groups such ~~s with cyanogen bromide (CNBr) linkage through a spacer, or by attachment vi a glycosylations in the Fc region of the antibody using hydrazine beads (available; commercially, e.g., from Bio-Rad, Hercules, California). For non-covalent attachments to the support, addition of an affinity tag sequence to the antibody can be used such as GST (Smith, D.B., and Johnson, K.S. Gene 67:31 (1988)); polyhistidines (Hochuli, E. et al., J.
Chromatog. 411:77 ( 1987)); or biotin. Alternatively, an indirect coupling agent such as Protein A or Protein G (available; commercially, e.g., from Sigma Chemical Co., St. Louis, Missouri) which binds to the Fc region of antibodies may be attached to the solid support and the .antibodies of the invention attached thereto by simply incubating the antibodies with the solid support containing the immobilized Protein A or Protein G. Such affinity tags may be also used for the reversible attachment of the antibodies of th.e present invention to the support.
Transgenic Animals The present invention is also directed to transgenic animals comprising one or more of the above-described mammalian RADI nucleic acid molecules.
Preferred such transgenic animals may be nnice comprising one or more of the above-described HRADI or~MRADl isolated nucleic acid molecules, or altered HRADI orMRADl genes that include mutations rendering the genes functionally inactive. Particularly preferred are those tranagenic animals comprising mutants, derivatives or variants of HRADI or MRf(Dl nucleic acid molecules which comprise deletion of one or more nucleotides from, or insertion or substitution of one or more nucleotides into, the corresponding wildtype HRADI or MRADI
gene such as those having the nucleotide sequences set forth in SEQ ID NO:1 or SEQ ID N0:3, respectively.
Transgenic animals comprising the nucleic acid molecules of the invention may be produced according to methods that are well-known in the art and that will be familiar to the skilled artisan (see, e.g., WO 90/05188;
Hammer, R.E., et al., J. Animal Sci. 63:269-278 (1986); Pursel, V.G., et al., J.
Reprod.
Fert. Suppl. 40:235-245 (1995); Houdebine, L.-M., J. Biotechnol. 34:269-287 (1994); Hammer, R.E., et al., Nature 315:680-683 (1985); Mortensen, R.M., et al., Mol. Cell. Biol. 13:2391-2395 (1992);. Deng, C., et al. , Cell 82:675-684 (1995); and Murakami, T., et al., Devel. Gen. 10:393-401 (1989), the disclosures of all of which are incorporated herein by reference in their entireties). In particular, transgenic animals and cell lines comprising the nucleic acid molecules of the invention may be producE;d as described in detail below in Examples 4 and 5.
Uses The isolated mammalian RADl nucleic acid molecules, polypeptides and antibodies of the invention are useful in a variety of methods, for example in industrial, clinical and research settings. Included among these uses are the use of the present nucleic acid molecules or antibodies in the detection of mammalian RADl expression or production by isolated calls or tissues, or by cells and tissues in an animal, for example for use in certain diagnostic assays as detailed below.
In addition, the nucleic acid molecules, antisense oligonucleotides and ribozymes of the invention may be used in therapeutic methods for treating an individual that is predisposed to or suffering from a disorder (such as a cancer) characterized by increased sensitivity to DNA-damaging agents. Analogously, the present nucleic acid molecules, antisense oligonuclc;otides and ribozymes may be used in methods of inducing sensitivity to DNA-damaging agents in cells or tissues (for example cancer cells or tumors) that are; relatively resistant to such agents.
Diagnostic and Prognostic Uses The present invention also provides methods of determining the sensitivity of a cell, tissue or animal to the effects of DNA-damaging agents.
As used herein, a "DNA-damaging agent" nneans any physical, biological or chemical composition or force that acts to alter the structure (reversibly or irreversibly) of one or more DNA molecules in a cell, tissue or animal upon which the agent acts. Such DNA-damaging agents may include, for example, chemicals (including carcinogens, teratogens, mutagens or the like), radiation (including ionizing radiation (e.g., 'y- or X-radiation) and non-ionizing radiation such as photoradiation (e.g., ultraviolet (UV) radiation) or electromagnetic radiation (e.g., microwave, radio or electromagnetic field radiation)), and other agents which cause damage to DNA. Such damage may be in any form which causes the structure of the DNA molecule to be altered from its normal or wildtype state in the untreated cell, tissue or animal, including without limitation inducing cross-linking of adj acent or juxtaposed nucleotide bases (e.g., thymidine dimerization), inducing breaks in the sugar-phosphate backbone, or inducing nucleotide tautomerization or isomerization.
It is believed that certain tissues in animals (particularly mammals) that are sensitive to DNA-damaging agents (e~.g., an animal suffering from or predisposed to a cancer) express significantly decreased levels of functional mammalian RADIp protein and/or mRNA encoding a RADIp protein when compared to a corresponding "standard" animal, i.e., an animal of the same species that is less sensitive to DNA-damaging agents. Further, it is believed that decreased levels of the RAD 1 protein can he detected in certain tissues, cells and/or body fluids (e.g., sera, plasma, urine, and spinal fluid) from animals that are sensitive to DNA-damaging agents, when compared to samples from animals of the same species that are less sensitive to such agents. Thus, the invention provides methods useful during diagnosis of the sensitivity of an animal to a DNA-damaging agent, which involve assaying the expression level of the gene encoding a mammalian RADlp protein in tissues, cells or body fluid from the animal and comparing this gene expression level with a standard mammalian RADl gene expression level, whereby a dii~erence (particularly a decrease) in RADl gene expression level compared to that of the standard is indicative of increased sensitivity to a DNA-damaging agent. Such methods for diagnosing the sensitivity of a human preferably involve assaying the level of expression of an HRADl gene in the human. Analogously, such methods for diagnosing the sensitivity of a mouse preferably involve assaying the level of expression of an MRADl gene in the mouse.
Methods according to this aspect of the invention may comprise one or more steps. For example, one such method of the invention may comprise:
(a) obtaining a first biological saanple from a first animal to be tested for its sensitivity to a DNA-damaging agent and a second biological sample from a second animal of known sensitivity to the DNA-damaging agent;

(b) determining the level of expression of a mammalian RADI gene in the first and second biological samples; and (c) comparing the level of mammalian RADl gene expression in the first biological sample to the level of mammalian RADI gene expression in the second biological sample, wherein a difference, particularly a decrease, in the level of mammalian RADI gene expression in the first biological sample relative to that of the second biological sample indicsates a higher level of sensitivity to a DNA-damaging agent in the first animal rc;lative to that of the second animal.
Another such method of the invention may comprise:
(a) obtaining a first biological sample from a first animal to be tested for its sensitivity to a DNA-damaging agent and a second biological sample from a second animal of known sensitivity to the DNA-damaging agent;
(b) determining the level or activity of a mammalian RADlp polypeptide in the first and second biological samples; and (c) comparing the level or activity of a mammalian RADlp polypeptide in the first biological sample to the level or activity of a mammalian RADIp polypeptide in the second biologiical sample, wherein a difference, particularly a decrease, in the level or activity of a mammalian RADlp polypeptide in the first biological sample relative to that of the second biological sample indicates a higher level of sensitivity to a DNA-damaging agent in the first animal relative to that of the second animal.
In practice, since the sensitivity of the first animal to a DNA-damaging agent may be estimated by determining the level of mammalian RAD1 gene expression, or the level or activity of mammalian RAD lp polypeptide, in the first biological sample relative to that in a second biological sample from a second animal of known sensitivity, the second biological sample thus serves as a control (positive or negative) in the present diagnostic methods. In some assays, it may be preferable to use two or more such control samples obtained from animals of varying sensitivities to a DNA-damaging agent to establish a "standard curve"
of sensitivity versus mammalian RADI gene expression; this standard curve may then be used to interpolate or extrapolate thc; sensitivity of the first animal to the DNA-damaging agent by plotting the mammalian RADI gene expression level in the first biological sample on the standard curve.
Where a diagnosis has already been made according to conventional methods, the present invention is useful ass a prognostic indicator, whereby animals exhibiting decreased mammalian h'ADI gene expression or decreased levels or activity of mammalian RADIp polypeptide will experience a worse clinical outcome relative to animals expressing the gene at a higher level.
By "determining the level of expression of a mammalian RADI gene" is intended qualitatively or quantitatively measuring or estimating the level of the mammalian RADIp polypeptide (such s~s an HRADlp or an MRADlp polypeptide) or the level of the mRNA encoding the mammalian RADIp polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the RAD 1 p polypeptide level o~r mRNA level in a second biological sample). Analogously, by "determining the level or activity of a mammalian RAD 1 p polypeptide" is intended qualitatively or quantitatively measuring or estimating the amount or biological activity of the mammalian RADlp polypeptide (such as an HRADlp or an MRADIp polypeptide), in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g. , by comparing to the RAD 1 p polypeptide level or mRNA level in a second biological sample).
Biological samples assayable accordling to these methods may be derived from any animal, preferably a mammal such as a human, ape, monkey, rat or mouse, and most preferably a human, and lnay comprise cells, tissues, organs or bodily fluids, or extracts thereof. The first and second biological samples may, but need not necessarily, be derived from two individuals of the same species.
In an alternative embodiment of the invention, the first and second biological samples may be derived from the same individual. Cells that are preferably used in these methods include primary cells derived from tissue biopsies or other means of obtaining primary cells from the animal, particularly epithelial cells, connective tissue cells (e.g., fibroblasts or adipose cells), neuronal cells, endothelial cells, leukocytes, germ cells (oocytes or spermatocytes), bone cells (osteocytes, osteoblasts or osteoclasts), .cartilage cells (chondrocytes or chondroblasts), stromal cells of the bone marrow, and other cell types that may express a mammalian RADI gene. Tissues useful in the present methods include, but are not limited to, ovarian, prostate, heart, placenta, pancreas, liver, spleen, lung, breast, neuronal, epithelial, bone marrow and umbilical tissues.
Mammalian body fluids that may be assayed according to the present methods include sera, plasma, urine, synovial fluid and spinal fluid. Methods for obtaining tissue biopsies, cell suspensions and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
To determine the level of mammalian RADl gene expression in a biological sample by measuring RADI mRN.A, it is preferable to first isolate total RNA from the sample. Total RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal.
Biochem. 162:156-159 (1987). Levels of mRNA encoding the mammalian RAD 1 protein are then assayed using any appropriate method. These include northern blot analysis, S 1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in comb ination with the ligase chain reaction (RT-LCR).
Northern blot analysis can be performed as described in Harada et al., Cell 63:303-312 ( 1990). Briefly, total RNA is prepared from a biological sample as described above. For the northern blot, the; RNA is denatured in an appropriate buffer (such as glyoxaUdimethyl sulfoxide/sodium phosphate buffer), subj ected to agarose gel electrophoresis, and transferred onto either a nitrocellulose filter or a nylon membrane. After the RNAs have been linked to the nitrocellulose filter by baking the filter in vacuo, or to the nylon membrane using a UV
crosslinker, the filter is prehybridized in a solution containing formamide, SSC, Denhardt's solution, denatured salmon sperni, SDS, and sodium phosphate buffer.
Mammalian RADIp polypeptide-encoding nucleic acid molecules or cDNAs, such as those of the invention prepared as described above, may be labeled -$1-according to any appropriate method (such as the 32P-multiprimed DNA labeling system (Amersham)) and used as hybridization probes. Preferred such probes are at least 10-1$ basepairs in length. After hybridization overnight, the filter is washed and exposed to x-ray film.
$ S1 mapping can be performed as described in Fujita et al., Cell 49:3$7-367 ( 1987). To prepare probe DNA for use in S 1 mapping, the sense strand of the above-described mammalian RADl cD:~IA may be used as a template to synthesize labeled RADI antisense DNA, lby methods known in the art and described in more detail below. The RADl antisense DNA can then be digested using an appropriate restriction endonucleasc; to generate further DNA probes of a desired length. Such antisense probes are useful for visualizing protected bands corresponding to the target mRNA (i. e., mRNA encoding a mammalian RAD 1 p polypeptide). Northern blot analysis can be performed as described above.
Alternatively, levels of mRNA encoding a mammalian RAD 1 protein may be assayed using the RT-PCR method described in Makino et al., Technique 2:29$-301 (1990). By this method, the radioactivities of the "amplicons" in the polyacrylamide gel bands are linearly related to the initial concentration of the taxget mRNA. Briefly, this method involves adding total RNA isolated from a biological sample in a reaction mixture containing a RT
primer and appropriate buffer. After incubating for primer annealing, the mixture can be supplemented with a RT buffer, dNTP s, DTT, RNa.se inhibitor and reverse transcriptase. After incubation to achieve reverse transcription of the RNA, the RT products are then subj ect to PCR using labeled primers. Alternatively, rather than labeling the primers, a labeled dNTP can be included in the PCR reaction mixture. PCR amplification can be performed in a DNA thermal cycler according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction mixture is electrophoresed on an agarose gel. After drying the gel, the radioactivity of the appropriate bands (corresponding to the mRNA encoding a mammalian RADlp polypeptide) is quantified using an imaging analyzer. RT' and PCR reaction ingredients and conditions, reagent and gel concentrations, and labeling methods are well known in the art. Variations on the RT-PCR method will be apparent to the skilled artisan.
Any set of oligonucleotide primers which will amplify reverse-transcribed target mRNA can be used and can be designed as described in the Examples below.
Assaying mammalian RADIp polypeptide levels in a biological sample can occur using any art-known method. Preferred for assaying mammalian RADIp polypeptide levels in a biological sample are antibody-based techniques.
For example, RADIp polypeptide expression in tissues, cells or body fluids can be studied with classical immunohistological or immunocytological methods. In these, specific recognition is provided by a primary antibody (polyclonal or monoclonal) but a secondary detection system can utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a result, an immunohistological or immunocytological staining of the tissue section or cell suspension for pathological examination is obtained. Tissues and cells can also be extracted, e.g. , with urea and neutral detergent, for the liberation of mammalian RAD 1 p polypeptide for western blot or dot/slot assay (Jalkanen, M., et al., J. Cell Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell Biol. 105:3087-3096 (1987)).
In this technique, which is based on the use of cationic or anionic solid phases, quantitation of mammalian RAD 1 p polypeptide can be accomplished using isolated RADlp polypeptide or fragments thereof obtained according to the present invention as a standard. This technique can also be applied to body fluids. With these samples, a molar concentration of mammalian RADlp polypeptide will aid to set standard values of RADIp polypeptide content for different body fluids, like serum, plasma, urine, spinal fluid, etc. The normal appearance of RAD lp polypeptide amounts can be set using values from healthy individuals (e.g., those individuals that are less sensitive to DNA-damaging agents), which can then be compared to those obtained from a test subj ect.
Other antibody-based methods useful for detecting mammalian RADI
gene expression include immunoassays, such as ELISA and RIA as will be familiar to one of ordinary skill. For example, a mammalian RADlp polypeptide-specific antibody, such as that obtained by the methods of the present invention, can be used both as an immunoadsorbent and as an enzyme-labeled proba to detect and quantify the RADIp polypeptide. The amount of RADIp polypeptide present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
Such an ELISA for detecting a tumor antigen is described in Iacobelli et al., Breast Cancer Research and Treatment 11:19-30 (1988). In another ELISA
assay, two distinct specific monoclonal antibodies can be used to detect mammalian RADlp polypeptide in a tissue, cell, body fluid or extract thereof, in a modification of the "sandwich" assay described above. In this assay, one of the antibodies is used ass the immunoadsorbent and the other as the enzyme-labeled probe.
The above techniques may be conducted essentially as "one-step" or "two-step" assays. A "one-step" assay involves contacting a mammalian RAD 1 p polypeptide with immobilized antibody and, without washing, contacting the mixture with the labeled antibody. A "two-step" assay involves washing before contacting the mixture with the labeled anti body. Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
These methods of the invention are pairticularly useful in several diagnostic applications. For example, such methods may be used to identify an individual animal, particularly a maunmal such as a human, that is at risk of or predisposed to developing a disorder, such as a cancer, that is characterized by increased sensitivity of the cells of the inClividual to a DNA-damaging agent relative to cells of an individual that is not so predisposed to or at risk of developing the disorder. In addition, the pre;;ent methods may be used to identify an individual, particularly a maunmal suclh as a human, that is sensitive to radiation such ass those ionizing and non-ioni:;ing radiation types described above.
Use of these methods of the invention to diagnose the sensitivity of such individuals to DNA-damaging agents or radiation may be useful in the clinical setting, for example, to identify individuals at a higher risk of developing cancer than individuals in the general population. These methods may also dictate the choice of therapy in such individuals; for example, the use of radiation or chemotherapy protocols, which would leadl to increased side effects in such individuals due to their sensitivity to DNA-damaging agents, might be discouraged in such individuals. The pre.:ent diagnostic methods may also facilitate therapeutic approaches, such as those described below, to preventing the development, or delaying the onset, of the above-described disorders in such individuals.
Therapeutic Uses The isolated nucleic acid molecules and polypeptides of the invention may also be used therapeutically, for example; in treating or preventing a disorder, characterized by increased sensitivity to a DANA-damaging agent, in an animal, particularly a mammal such as a human or mouse, predisposed thereto or suffering therefrom. In such approaches, the goal of the therapy is to delay or prevent the development of the disorder, and/or to cure or induce a remission of the disorder, and/or to decrease or minimize the side effects of other therapeutic regimens.
Methods according to this aspect of the invention may comprise one or more steps which allow the clinician to achieve the above-described therapeutic goals. A preferred such method may comprise:
(a) obtaining a first biological sample from a first animal and a second biological sample from a second animal of known sensitivity to a DNA-damaging agent;
(b) determining the level of mammalian RADI gene expression in the first biological sample relative to that of the; second biological sample; and (c) treating a first animal, in which the the level of mammalian RADI
gene expression in the first biological sample is different (i.e., higher or lower, particularly lower) than that of the second biological sample, by a technique that cures, delays or prevents the development of, or induces remission of, the disorder.

In the present methods, the determination of the level of mammalian RADI gene expression in the first biological ;ample relative to that of the second biological sample is preferably performed as described above, by measuring the level or activity of mammalian RAD 1 p polyp~eptide and/or mRNA production in the samples. In this way, animals that are sensitive to DNA-damaging agents, or predisposed to, at risk for, or suffering from a disorder (such as a cancer) characterized by increased sensitivity to such agents, may be identified and aggressively and/or proactively treated.
Conventional Therapies. In a first such aspect of the invention, the technique that cures, delays or prevents the development of, or induces remission of, the disorder may be a conventional therapeutic approach commonly used in treating or preventing such disorders. Such approaches include, without limitation, aggressive chemotherapy (e.g., wiith one or more approved chemicals or other drugs that cure, delay or prevent the development of, or induce remission of, the disorder), radiation therapy (including; focal and whole-body irradiation), surgery (including focal and radical invasive or non-invasive surgical removal of affected tissues and/or cells, with or without removal of surrounding uninvolved tissues and/or cells). More preferably, a combination of such approaches is used, particularly a combination of chemotherapy and radiation therapy (see Sporn, M.B., Lancet 347:1377-1381 (1996)). Appropriate chemotherapeutic agents, radiotherapeutic agents and surgical approaches, and combinations thereof, are well-known in the art and will be familiar to the skilled clinician.
Of course, one significant advantage of the diagnostic methods of the present invention is the identification of these individuals in which the level or activity of RADI mRNA or RAD 1 p polype:ptide is lower than in a standard or control biological sample. As noted above, these individuals with lower RADI
expression will be expected to be more sensitive to treatment with DNA-damaging agents such as radiation and many chemotherapeutic agents. Thus, the diagnostic methods of the invention may be used to indicate alternative therapeutic approaches in such individuals, i:or example the use of a combination of surgery and chemotherapy with agents that do not induce DNA damage (e.g., taxol or taxanes).

Antisense Oligonucleotides. In an alternative therapeutic aspect of the invention, the technique that cures, delays or prevents the development of, or induces remission of, the disorder in the animal may comprise introducing into the animal a composition comprising one or more antisense oligonucleotides that are designed to interact with the mammali;m RADI mRNA produced by the affected cells or tissues, thereby preventing or delaying expression of the mammalian RAD 1 p polypeptide and thus development and/or progression of the disorder. The invention thus further provides such mammalian RADI antisense nucleic acid molecules (such as oligonucleotides), such as HRADl or MRADl antisense nucleic acid molecules which may interact with HRADI (in the case of HRADI antisense molecules) or MRADl (in the case of MRADI antisense molecules). As used herein, the terms "an~~isense nucleic acid molecule" and "antisense oligonucleotide" (which may be used interchangeably) mean a DNA
or RNA molecule, or a derivative thereof, containing a nucleic sequence which is complementary to that of a specific mRIVA. An antisense oligonucleotide binds to the complementary sequence in a specific mRNA and inhibits translation, and/or negatively affects the stability, of the mRNA.
Alternatively, an antisense oligonucleotide can bind to t:he complementary sequence on a specific DNA molecule (such as a mammalian RADl DNA molecule) and inhibit transcription of that DNA molecule.
There are many known derivatives of such DNA and RNA molecules (see, e.g., U.S. Patent Nos. 5,602,240, 5,596,091, 5,506,212, 5,521,302, 5,541,307, 5,510,476, 5,514,787, 5,543,50 ~~, 5,512,438, 5,510,239, S,S
14,577, 5,519,134, 5,554,746, 5,276,019, 5,286,717 and 5,264,423; see also WO
96/35706 (IJ.S. Application No. 08/438,97-'i), WO 96/32474 (L1.S. Application No. 08/420,672), WO 96/29337 (U.S. Application No. 08/409,169) (thiono triester modified antisense oligodeoxynu~cleotide phosphorothioates), WO
94/17093 (U.S. ApplicationNo. 07/939,262) (oligonucleotide alkylphosphonates and alkylphosphothioates), WO 94/08004 (U.S. Application No. 07/958,135) (oligonucleotide phosphothioates, methyl phosphates, phosphoramidates, dithioates, bridged phosphorothioates, bridge phosphoramidates, sulfones, sulfates, ketones, phosphate esters and phosphorobutylamines (van der Krol et al., Biotech. 6:958-976 (1988); Uhlmann et al., Chem. Rev. 90:542-585 (1990)), WO 94/02499 (U.S. Application No. 07/919,967) (oligonucleotide alkylphosphonothioates and arylphosphonothioates), and WO 92/20697 (U.S.
Application No. 07/698,568) (3'-end capped oligonucleotides), the disclosures of which are incorporated herein by reference in their entireties). Particular mammalian RADI antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos;
see Cohen, J., Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, Boca Raton, Florida: CRC Press (1989)). S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention may be prepared by treatment of the corresponding O-oligos with 3H 1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur transfer reagent (see Iyer et al., J. Org. Chem. 55:4693-4698 ( 1990);
and Iyer et al., J. Am. Chem. Soc. 112:1253-1254 (1990)).
Antisense oligonucleotides have been described as naturally occurnng biological inhibitors of gene expression in both prokaryotes (Mizuno et al., Proc.
Natl. Acad. Sci. USA 81:1966-1970 ( 1984)) and eukaryotes (Heywood, Nucl.
Acids Res. 14:6771-6772 (1986)), and these. sequences presumably function by hybridizing to complementary mRNA sequences, resulting in hybridization arrest of translation (Paterson, et al., Proc. Natl. Acad. Sci. USA 74:4370-4374 (1987)).
Antisense oligonucleotides are short, usually synthetic, DNA or RNA
polynucleotide molecules formulated to be complementary to a specific gene or RNA message. Through the binding of these oligomers to a target DNA or mRNA sequence, transcription or translation, respectively, of the gene can be selectively blocked and the disease process ,generated by that gene can be halted (see, for example, Cohen, J., Oligodeoxynucleotides, Antisenselnhibitors ofGene Expression, Boca Raton, Florida: CRC Press (1989)). The cytoplasmic location of mRNA provides a target considered to~ be readily accessible to antisense oligodeoxynucleotides entering the cell; hence much of the work in the field has focused on RNA as a target. Currently, the use of antisense oligodeoxynucleotides provides a useful tool for exploring regulation of gene -$ 8-expression in vitro and in tissue culture (Rotlaenberg, et al., J. Natl.
Cancer Inst.
81:1539-1544 (1989)).
Antisense therapy comprises the administration of exogenous oligonucleotides which bind to a target polynucleotide located within the cells, which may be performed in vitro or in vivo. For example, antisense oligonucleotides may be administered systemically for anticancer therapy (see U.S. Patent No. 5,087,617). As described above and in detail in the Examples below, defects in cell cycle checkpoint control genes (such as mammalian RADl genes) are thought to be involved in the genesis of certain disorders in humans, such as cancers. Thus, HRADI and MRADI antisense oligonucleotides of the present invention may be active in the therapeutic methods of the present invention as a technique that cures, delays or prevents the development of such a disorder. In particular, such methods function by rendering the affected cells or tissues more sensitive to DNA-damaging agents (such as radiation or chemicals), thereby increasing the efficacy of the conventional therapeutic approaches described in detail above.
According to this aspect of the invention, the mammalian RADI antisense oligonucleotides may be used in combination with one or more of the above-described conventional therapies in at lea;ct two ways. In a first aspect, the mammalian RADl antisense oligonucleotide s of the invention may be introduced in a disseminated fashion into the animal to be treated (e.g., by intravenous or oral administration, or other systemic means of introduction of therapeutic agents that will be familiar to the skilled clinician), and the conventional therapy may be localized to one or more particular sites to be treated (e.g., targeted radiation exposure of one or more tumor sites). In a related aspect, the mammalian RADI
antisense oligonucleotides of the invention may be introduced in a localized fashion into the animal to be treated (e.g., by localized injection, intranasal or intraocular administration, etc.), and the conventional therapy used may be of a more disseminated or generalized type (e.g., conventional chemotherapy).
As discussed above, the invention relates to the amino acid and nucleotide sequences for mammalian RADI genes such as HRADI and MRADI. Thus, the antisense oligonucleotides of the invention preferably have a nucleic acid sequence that is complementary to substantially all or a portion of the mRNA
which may be transcribed from SEQ ID NO:1 (in the case of HRADI antisense molecules) or SEQ ID N0:3 (in the case of MRADl antisense molecules), or to fragments thereof including those sequence.. encoding epitope-bearing regions of HRADIp or MRADlp polypeptides as described above. In one preferred embodiment, the antisense oligonucleotide;s are DNA molecules, or DNA
phosphorothioate molecules, or derivatives thereof. In another preferred embodiment, the antisense oligonucleotides are 15- to 40-mers, more preferably 15- to 30-mers.
Examples of preferred nucleotide sequences for HRADl antisense oligonucleotides include, but are not limited to:
CGTCCACTGCGCATTCGGCCCCGAGGGATG (SEQ ID NO:S) GGGTCAGAAGGGGCAT (SEQ ID N0:6) GGCTGTACTGATCATCCTCG (SEQ ID ~V0:7) TAACGTTGTCAAGGCTGGCC (SEQ ID N0:8) GCCTGAATAAAAGCATTTGCTTGCACACAC (SEQ ID N0:9) GGCTTGTCAGGAGACATGGT (SEQ ID NO:10) Examples of preferred nucleotide sequences for MRADl antisense oligonucleotides include, but are not limited to:
GGATGGTCCACGGTACCGTCGGCTCC;GAGA (SEQ ID NO:11 ) TACTGGGTTAGGAGA (SEQ ID N0:12) ACTGTTCGTACTCCTCTTCA (SEQ ID N0:13) AAGGCTGGCCACTAAGCAGT (SEQ ID N0:14) AACCTTGGTAACACATCCGAAGCGC~~GTCA (SEQ ID NO:15) ACTGTAAGGAGAGGAAGCCT (SEQ II) N0:16) Other nucleic acid molecules and nucleotide sequences thereof that may be useful as HRADl and MRADI antisense oligonucleotides according to the invention will be apparent to one of ordinary skill, based on the nucleotide sequences of the HRADl cDNA (SEQ ID 1V0:1) and MRADl cDNA (SEQ ID
N0:3) and the guidance provided herein.

Also provided for use in the pre:>ent methods are pharmaceutical compositions comprising a therapeutically effective amount of one or more antisense oligonucleotides and a pharmaceutically acceptable carrier or excipient therefor. In one embodiment, a single mammalian RADI antisense oligonucleotide is utilized. In another embodiment, two or more mammalian RADl antisense oligonucleotides are utilized which are complementary to adj acent regions of the mammalian RADl DT(A or the mammalian RADI mRNA
that is transcribed from the mammalian RADA nucleic acid molecules described above. Administration of two or more mammalian RADI antisense oligonucleotides which are complementary to adjacent regions of the DNA or corresponding mRNA may allow for more efficient inhibition of RADI genomic transcription or RADI mRNA translation, resulting in more effective inhibition of the expression of mammalian RADI , thereby inducing a prevention or delaying of the disorder. In a related aspect of the invention, the mammalian RADI
antisense oligonucleotides or composition;. comprising the mamalian RADI
antisense oligonucleotides of the invention may be used in combination with one or more of the above conventional therapies, to provide a more rapid therapeutic or preventative course of treatment.
Preferably, the mammalian RAM antisense oligonucleotide is coadministered in a composition further comprising one or more agents that enhance the uptake of the antisense moleculf~ by the affected cells. For example, the one or more mammalian RADI antisense; oligonucleotides may be combined with a pharmaceutically acceptable carrier or excipient therefor, as described below. The present antisense oligonucleotides may also be administered in a composition with one or more lipophilic cationic compounds or carriers, which may be in the form of liposomes. The use of liposomes to introduce polynucleotides into cells is disclosed, i:or example, in U.S. Patent Nos.
4,897,355 and 4,394,448. See also U.S. Patent Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567, 4,247,41:1, 4,814,270 for general methods of preparing liposomes comprising biological materials. In addition, the present antisense oligonucleotides may be combined with alternative lipophilic carriers such as any one of a number of sterols including cholesterol, cholate and deoxycholic acid. A preferred sterol is cholE;sterol.
The mammalian XADI antisense oligonucleotides of the invention additionally may be conjugated to a peptide that is ingested by the affected cells.
Examples of useful peptides include peptide: hormones, antigens or antibodies, and peptide toxins. By choosing a peptide that is selectively taken up by the neoplastic cells, specific delivery of the ant:isense agent may be effected.
The present antisense oligonucleotides may be activated by formation of an activated aminoalkyl derivative at the 5'OH group. ':~Che peptide of choice may then be covalently attached to the activated mammalian RADl antisense oligonucleotide via an amino- and sulfhydryl-reactive heterobifunctional reagent. The latter is bound to a cysteine residue present in the pptide. Upon exposure of cells to the mammalian RADl antisense oligonucleotide bound to the peptide, the peptidyl antisense agent is endocytosed and the anti;sense oligonucleotide binds to the target mammalian RADl mRNA to inhibit translation (see U.S. Patent No.
5,525,465; see also EP 0 263 740).
Antisense oligonucleotides can be prepared which are designed to interfere with transcription of the mammalian RADI gene (such as an HRADl or an MRADI gene) by binding transcribed regions of duplex DNA (including introns, exons, or both) and forming triple helices (see U. S. Patent Nos.
5,399,676, 5,594,121 and 5,591,607; WO 96/35706 (U.S. Application No.
08/438,975); WO 96/32474 (LJ.S. Application No. 08/420,672); WO 94/17091 (U.S. Application No.07/938,000); WO 94/01550 (U.S. Application No.
07/909,069); and WO 92/10590 (U.S. .Application No. 07/625,680), the disclosures of which are incorporated herein by reference in their entireties).
Preferred antisense oligonucleotides :for triple helix formation are oligonucleotides which have inverted polarities for at least two regions of the oligonucleotide (Id.). Such oligonucleotides may comprise tandem sequences of opposite polarity such as 3'---5'-L-5'---3', or :5'---3'-L-3'---5', wherein L
represents a 0-10 base oligonucleotide linkage between oligonucleotides. The inverted polarity form stabilizes single-stranded oligonucleotides to exonuclease degradation (Id.).

The antisense oligonucleotides of the. present invention may be prepared according to any of the methods that are well known to those of ordinary skill in the art, including methods of solid phase; synthesis and other methods as disclosed in the publications, patents and patent applications cited herein.
Riborymes. In still another approach to the therapeutic methods of the invention, the technique that cures, delays or prevents the development of, or induces remission of, the disorder in the animal may comprise introducing into the animal a composition comprising one or :more ribozymes that are designed to interact with the mammalian RADI mRNA yroduced by the affected cells and/or tissues, thereby preventing or delaying expression of the RADlp polypeptide.
Thus, the invention is also directed to ribozymes comprising a target sequence which is complementary to a mammalian RADI DNA sequence, such as an HRADI or an MRADI DNA sequence, or to a mammalian RADl mRNA
sequence that is transcribed from the above-described nucleic acid molecules (such as the HRADI nucleotide sequence dpicted in Figure 1 (SEQ ID NO:1) or the MRADl nucleotide sequence depicted in Figure 2 (SEQ ID N0:3)). The invention is also directed to pharmaceutical compositions comprising such ribozymes and a pharmaceutically acceptable earner or excipient therefor.
Ribozymes, which provide an altf;rnative method to inhibit mRNA
function, may be RNA enzymes, self splicing RNAs or self cleaving RNAs (Cech, T., et al., J. Biol. Chem. 267:17479-17482 (1992)). It is possible to construct ribozymes de novo which have an endonuclease activity directed in trans to a certain target sequence. Since these ribozymes can act on various sequences, ribozymes can be designed for virtually any RNA substrate and thus are very flexible tools for inhibiting the expression of specific genes. For example, a ribozyme against chloramphenicol acetyltransferase mRNA has been successfully constructed (Haseloff et al., Nature 334:585-591 (1988);
Uhlenbeck et al., Nature 328:596-600 (1987)). This ribozyme contains three structural domains: 1 ) a highly conserved region of nucleotides which flanks the cleavage site in the 5' direction; 2) the highly conserved sequences contained in naturally occurring cleavage domains of ribozymes, f arming a base-paired stem; and 3) the regions which flank the cleavage site on both sides and ensure the exact arrangement of the ribozyme in relation to the cleavage site and the cohesion of the substrate and enzyme. RNA enzymes constructed according to this model have already proven suitable in vitro for the ;specific cleaving of RNA
sequences (Haseloff et al., Nature 334:585-591 (1988)) such as the antisense oligonucleotides described above.
Alternatively, hairpin ribozymes may be used in which the active site is derived fiom the minus strand of the satellite RNA of tobacco ring spot virus (Hamper et al., Biochemistry 28:4929-4933 (1989)). For example, hairpin ribozyrnes have been designed which cleave human immunodeficiency virus type 1 RNA (Ojwang et al.) Proc. Natl. Acad Sci. I~SA 89:10802-10806 (1992)). Other self cleaving RNA activities are associated with hepatitis delta virus (Kuo et al., Virol. 62:4429-4444 (1988)). See also U.S. :Patent No. 5,574,143, the disclosure of which is incorporated by reference herein) for methods of preparing and using ribozymes.
Preferably, the mammalian RADI ,ribozyme molecules of the present invention are designed based upon the chloramphenicol acetyltransferase ribozyme or hairpin ribozymes, described above. Alternatively, the present ribozyme molecules may be designed as described in WO 92/07065, which discloses catalytically active ribozyme constructions having increased stability against chemical and enzymatic degradation that are therefore useful as therapeutic agents.
Also provided by the present invention are pharmaceutical compositions comprising an effective amount of at least one ribozyme of the invention in combination with a pharmaceutically acceptable Garner or excipient therefor.
Preferably, the ribozyme is coadministe:red as a composition that further comprises one or more agents that enhance the uptake of the ribozyme by the affected cells. For example, the present ribozyme compositions may comprise one or more lipophilic Garners (such as cholesterol) or lipophilic cationic compounds which may be in the foam of liposomes, as described above for mammalian RADI antisense oligonucleotides.
Ribozyme therapy comprises the administration of exogenous ribozyme oligonucleotides which bind to a target polynucleotide located within the cells, which may be performed in vitro or in vivo. F'or example, as described above for antisense therapy, ribozymes may be administered systemically for anticancer therapy. As described above and in detail in the Examples below, defects in cell cycle checkpoint control genes (such as mammalian RADI genes) are thought to be involved in the genesis of certain disorders in humans, such as cancers.
Thus, HRADI and MRADl ribozymes of the present invention may be active in the therapeutic methods of the present invention as a technique that cures, delays or prevents the development of such a disorder. In particular, such methods function by rendering the affected cells o:r tissues more sensitive to DNA-damaging agents (such as radiation or chemicals), thereby increasing the efficacy of the conventional therapeutic approaches ~~s described.
According to this aspect of the invention, the mammalian RADI
ribozymes may be used in combination with one or more of the above-described conventional therapies in at least two ways. In a first aspect, the mammalian RADI ribozymes of the invention may be introduced in a disseminated fashion into the animal to be treated (e.g., by intravenous or oral administration, or other systemic means of introduction of therapeutic agents that will be familiar to the skilled clinician), and the conventional therapy may be localized to one or more particular sites to be treated (e.g., targeted radiation exposure of one or more tumor sites). In a related aspect, the m~unmalian RADl ribozymes of the invention may be introduced in a localized ifashion into the animal to be treated (e.g., by localized injection, intranasal or intraocular administration, etc.), and the conventional therapy used may be of a more disseminated or generalized type (e.g., conventional chemotherapy).
The antisense oligonucleotides, ribozymes and pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose of curing, delaying or preventing the development of, or inducing remission of, a disorder in an animal. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intra-peritoneal, transdermal, intrathecal or intracranial routes. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. For example, as much as 700 milligrams of antisense oligodeoxynucleotide has been administered intravenously to a patient over a course of 10 days (i. e., O.US mg/kg/hour) without signs of toxicity (Sterling, "Systemic Antisense Treatment Reported," Gen. EngNews 12(12):1, 28 ( 1992)).
While individual needs may vary, determination of optimal ranges of effective amounts of each component is within the ability of the clinician of ordinary skill.
In addition to administering the present antisense oligonucleotides or ribozymes as raw chemicals in solution, the therapeutic molecules may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the present antisense oligonucleotides or ribozymes.
Suitable formulations for parenteral administration include aqueous solutions of the present antisense oligonucleotides or rib~ozymes in water-soluble form, for example, as water-soluble salts. In addiition, suspensions of the active compounds as appropriate oily injection suspensions may be administered.
Suitable lipophilic solvents or vehicles includle fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ettryl oleate or triglycerides.
Aqueous inj ection suspensions may contain substances which increase the viscosity of the suspension; such substances may include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
Alternatively, the present antisense o ligonucleotides or ribozymes can be encoded by DNA constructs which are then administered in compositions comprising virions or vectors containing these DNA constructs. Preferably, the vectors or virions comprising the DNA constructs containing the present antisense oligonucleotides or ribozymes are incapable of replicating in vivo (see, e.g., WO 92/06693 (U.S. Application No. 07/602,135)). For example, such DNA
constructs may be administered using hepes-based viruses (U.S. Patent No.
5,082,670). In addition, the antisense olig;onucleotides and ribozyrnes of the invention may be encoded by RNA constmcts which are then administered in compositions comprising these constructs in the form of virions or vectors, such as retroviruses. The preparation of retroviral vectors is well known in the art (see, for example, Brown et al., "Retroviral Vectors," in DNA Cloning: A Practical Approach, Volume 3, Washington, D.C.: IRL Press (1987)).
Gene Therapy. In amother therapeutic;, aspect of the invention, the animal may be treated by introducing into the animal one or more of the isolated nucleic acid molecules of the invention comprising a polynucleotide encoding a mammalian RADIp polypeptide or a fragment thereof, particularly a polynucleotide that is 90% or 95% identical to one or more of the reference nucleotide sequences (such as that ofHRADl (SEQ ID NO:1) orMRADI (SEQ
ID N0:3) described above. This approach, known generically as "gene therapy,"
is designed to increase the level of maunmaliam RADI gene expression in the cells affected by or causing the disorder (such ass camcer or tumor cells) acnd thereby to cure, delay or prevent the development of, or induce remission of, the disorder by restoring cell cycle checkpoint control to the affected cells. Analogous gene therapy approaches have proven effective or to have promise in the treatment of certain maunmalian diseases such as cystic fibrosis (Drumm, M.L. et al., Cell 62:1227-1233 (1990); Gregory, R.J. et al., Nature 347:358-363 (1990); Rich, D.P. et al., Nature 347:358-363 (1990)), Gau~cher's disease (Sorge, J. et al., Proc.
Natl. Acad. Sci. USA 84:906-909 (1987); Fi~ilc, J.K. et al., Proc. Natl. Acad.
Sci.
USA 87:2334-2338 (1990)), certain forms ofhemophilia (Bontempo, F.A. et al., Blood 69:1721-1724 (1987); Palmer, T.D. et al., Blood 73:438-445 (1989);
Axekod, J.H. et al., Proc. Natl. Acad. Sci. USA 87:5173-5177 (1990);
Armentano, D. et al., Proc. Natl. Acad. Sci. USA 87:6141-6145 (1990)) arnd muscular dystrophy (Pau~tridge, T.A. et al., Nature 337:176-179 (1989); Law, P.K. et al., Lancet 336:114-115 (1990); Morgan, J.E. et al., J. Cell Biol.
111:2437-2449 (1990)), as well as in other b~eatments for certain cancers such as metastatic melanoma (Rosenberg, S.A. et al., Science 233:1318-1321 (1986);
Rosenberg, S.A. et al., N. Eng. J. Med. 319:1676-1680 (1988); Rosenberg, S.A.
et al., N. Eng. J. Med. 323:570-578 (1990)).
In a preferred such approach, one or more isolated nucleic acid molecules of the invention, comprising a polynucleotiide having a nucleotide sequence at least 90% or at least 95% identical to one or more of the above-described reference nucleotide sequences (such as the ~~RADI nucleotide sequence set forth in SEQ ID NO:1 or the MRADI nucleotide sE;quence set forth in SEQ ID N0:3), is introduced into or administered to the animal that is suffering from or predisposed to the disorder. Such isolated nucleic acid molecules may be incorporated into a vector or virion suitable for introducing the nucleic acid molecules into the cells or tissues of the animal to be treated, to form a transfection vector. Suitable vectors or viriions for this purpose include those derived from retroviruses, adenoviruses and adeno-associated viruses.
Alternatively, the nucleic acid molecules of the invention may be complexed into a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or with viral components (e.g., viral capsid proteins).
Techniques for the formation of vectors or virions comprising the mammalian RADl -encoding nucleic acid molecules are well-known in the art, and are generally described in "Working Toward Human Gene Therapy," Chapter 28 in RecombinantDNA, 2nd Ed., Watson, J.D. et al., eds., New York: Scientific American Books, pp. 567-581 (1992). In addition, general methods for construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, the disclosures of which are specifically incorporated herein by reference in their entirety. In one such general method, vectors comprising the isolated polynucleotides of the present invention are directly introduced into the cells or tissues of the affected animal, preferably by inj ection, inhalation, ingestion or introduction into a mucous membrane via solution; such an approach is generally referred to as "in vivo" gene therapy. Alternatively, cells, tissues or organs, particularly those containing cancer cells or tumors, may be removed from the affected animal and placed into culture according to methods that are well-known to one of ordinary skill in tlae art; the vectors comprising the mammalian RADI polynucleotides may then be introduced into these cells or tissues by any of the methods described generally above for introducing isolated polynucleotides into a cell or tissue, includin g viral infection or transfection, and, after a sufficient amount of time to allow incorporation of the RADI
polynucleotides, the cells or tissues may tlhen be re-inserted into the affected animal. Since the introduction of the mammalian RAD1 gene is performed outside of the body of the affected animal, this approach is generally referred to as "ex vivo" gene therapy.
For both in vivo and ex vivo gene therapy, the isolated mammalian RADI
polynucleotides of the invention may alternatively be operatively linked to a regulatory DNA sequence, which may be a mammalian RADI promoter or an enhancer, or a heterologous regulatory DN,A sequence such as a promoter or enhancer derived from a different gene, cell or organism, to form a genetic construct as described above. This genetic construct may then be inserted into a vector, which is then directly introduced into the affected animal in an in vivo gene therapy approach, e.g. , by intratumoral administration (i. e. , introduction of the nucleic acid molecule or vector directly into a tumor in an animal, for example by inj ection), or into the cells or tissues of the affected animal in an ex vivo approach. In another preferred embodiment, the genetic construct of the invention may be introduced into the cells or tissues of the animal, either in vivo or ex vivo, in a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or viral components (e.g ., viral capsid proteins; see WO
93/07283). These approaches result in increased production of RADIp polypeptide by the cells of the treated anirnal via (a) random insertion of the RADI gene into the host cell genome; or (b) incorporation of the RADl gene into the nucleus of the cells where it may exist as an extrachromosomal genetic element. General descriptions of such methods and approaches to gene therapy may be found, for example, in U.S. Patent No. 5,578,461; WO 94/12650; and WO 93/09222.
Regardless of the approach used, however, use of these methods of the present invention will result in the increased production of RADlp polypeptide by the cells and tissues of the treated animal, such that the disorder will be delayed or inhibited, or such that the disorder will go into remission or be cured.
Isolationlldentification of Cell Cycle Checkpoint Control Polypeptides The isolated mammalian RADI nucleic acid molecules, polypeptides and antibodies of the invention are also useful in methods for isolating additional cell cycle checkpoint control genes and polypeptides, particularly those that interact with the mammalian RADI nucleic acid molecules and polypeptides of the invention. Methods according to this aspect of the invention may comprise one or more steps, including, for example, contacting one or more HRADlp or MRADIp polypeptides of the invention with a sample (e.g., a cell lysate, a culture supernatant from cells expressing one. or more recombinant or natural cell cycle checkpoint control polypeptides, or the like) containing one or more cell cycle checkpoint control polypeptides that interact with HRADIp and/or MRADIp, under conditions (such as those described in detail in the Examples herein, and other conditions favoring protein:protein interaction that will be familiar to the ordinarily skilled artisan) favoring the interaction of the one or more cell cycle checkpoint control polypeptides with the HRADIp or MRADIp polypeptides of the invention. In certain. embodiments in this regard, the HRAD 1 p and/or MItAD 1 p polypeptides may be immobilized on a solid support and the sample containing the one or more cell cycle checkpoint control polypeptides may be passed over the solid phase containing the immobilized HRAD 1 p and/or MRAD 1 p polypeptides under conditions favoring the interaction (e.g., binding or association) of the cell cycle checkpoint control polypeptides with the immobilized HRAD lp and/or MRAD lp polypeptides. According to this embodiment, the desired cell cycle checkpoint control polypeptides may then be released from the immobilized HRAD 1 p and/or MRAD 1 p polypeptides, using, for example, elution by change in ionic strength, by competitive release (using, e.g. , additional HRAD 1 p or MRAD 1 p pol~,~peptide), by treatment with one or more chaeotropic agents (e.g., urea, guanidine salts, etc.), by treatment with high magnesium concentrations, etc., according to routine methods ofprotein isolation and recovery that are well-known in the art, thereby isolating the one or more cell cycle checkpoint control polypeptides. In an alternative such method, one or more cell cycle checkpoint control polypptides may be identified or isolated using the anti-HRAD 1 p and/or anti-MRAD'l p antibodies of the invention, by co-immunoprecipitation of HRAD 1 p or MRAI~ 1 p complexed with the one or more cell cycle checkpoint control polypeptides, as described in more detail in Examples 10-12 herein.

It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein may be made without departing from the scope of the invention or any embodiment thereof. Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.
Examples Materials and Methods The following materials and methods were generally used in the Examples, unless otherwise indicated.
PCR Amplification Oligonucleotide primers were synthesized on a Beckman Oligo 1000 DNA Synthesizer against various stretches of the expressed sequence tag (EST) (GenBank Accession number AA029300). Sequences of the oligonucleotide primers ("oligos") were as follows:
Oligo A: GGTACATGACCTTGCTCCTA'T (SEQ ID N0:17);
Oligo B: AGTTCCCACCTTGACTATCC (SEQ ID N0:18);
Oligo C: AGCCTCTGTTATCTGTCCGA (SEQ ID N0:19); and Oligo D: CTTGTAAGGTATCTATTCGG.ACA (SEQ ID N0:20).
Oligos A and C (SEQ ID NOs: 17 arid 19, respectively) were oriented in the forward direction while oligos B arid D (SEQ ID NOs: 18 and 20, respectively) were oriented in the reverse direction. These oligonucleotides were used to prime PCR reactions using HaCaT cDNA as a template. PCR
amplifications were carried out on an MJ R.esearch, Inc. programmable thermal cycler (model PTC-100). Reactions were done in a total volume of SO pl with ng HaCaT cDNA; 20 pmol of each oligonucleotide primer; 0.2 mM each dNTPs;
one unit of VENTTM DNA polymerase (New England BioLabs; Beverly, Massachusetts) in 1X Thermopol buffer (New England Biolabs). PCR was performed for 40 cycles, each consisting e~f one minute at 94°C and a final extension at 72°C, with a five minute pre-incubation at 95°C and a final extension at 72°C for five minutes. All possible primer pairs were used and 20 ~1 of the PCR products were resolved on a. 1 % agarose gel containing 40 ~,g ethidium bromide (EtBr) by electrophoresis for 35 minutes at 120 V, and visualized under ultraviolet light.
Plasmid Manipulations and Recovery Subcloning the two largest PCR products into a pBluescript (pBS KS-) plasmid at the SmaI site involved a two step blunt ligation. In a single 50 wl reaction of 1X T4 DNA Ligase buffer (Ne:w England Biolabs), 5 ~1 of PCR
product were phosphorylated at the 5' end with T4 polynucleotide kinase and 1 ~g of the vector was linearized with SmaI. This reaction was allowed to proceed for one hour at room temperature and the mixture was subsequently incubated overnight at 16°C with two units of T4 DNA Ligase.
After ligation, the DNA was extracted sequentially with equal volumes of phenol and 24:1 chloroform/isoamyl alcohol, and precipitated with two volumes of ethanol. Plasmids were transformed into electrocompetent XL 1-Blues using a BioRad Gene Pulser set at 25 wF, 2.5 kV and 200 ~2. White colonies were selected after overnight grovvth on LB agar plates (25 ml/plate) with ampicillin (Amp) at 0.05 mg/ml, ~S'.-Gal (800 pg/plate) and IPTG (1 ~ g/plate). Plasmids were recovered after overnight growth in LB/Amp. Inserts were sized using the appropriate restriction enzymes and subjected to electrophoresis on a 1 % agarose gel. Ma~;:i-preps for plasmid purification of successfully cloned products were performed using a QIAGEN-tip 500 (Qiagen, Inc.) according to the instructions of the m~mufacturer.
Subcloning of HRADI into pARTI
The HRADl open reading frame (ORF) was amplified with primers HRAD 1-5 (GGACGGTCGACATGCCCC'TTCTGACCCAA) (SEQ ID N0:21 ) and HRAD1-3 (ACGGATCCTCAAGACT'CAGATTCAGG) (SEQ ID N0:22), and blunt end ligated into the SmaI site of pART 1, to generate pART 1-HRAD 1.
Orientation of the inserts within pART 1 was determined by restriction digestion.
Screening of HaCaT cDNA
To isolate a full length human cDNA. clone, a screen of a HaCaT cDNA
~, phage library (obtained from David Beaclh, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) was performed. An overnight culture of E. coli BB4 was grown in NZCYM medium with 0.2% maltose and 1 mM MgS04. The primary screen involved a 30 minute incubation of 600 wl of this overnight culture with 106 ~, phage particles at 30°C. This mixture was then spread evenly (approximately 100 pl each) over 6 150mm~ NZCYM agar plates with 8 ml of NZCYM top agar for each plate. Double plaque lifts were performed for each plate onto nitrocellulose membranes (MicronSeparations, Inc.). Membranes were floated onto denaturing solution (1.5 M NaCI, 0.5 N NaOH) for four minutes, treated with neutralizing solution ( 1 M Tris,1.5 M NaCI, pH 7.4) for five minutes and then rinsed in 5X SSPE (0.75 M NaCI, 0.05 NaHzP04, 6.3 mM EDTA, pH 7.4). The membranes were allowed to ai:r dry and were baked under vacuum for 1.5 hours at 80°C in a National Appliance Company Model 5831 oven.
Membranes were probed with a random-primed probe generated by using the subcloned PCR product as a template and [a; 'ZP] dGTP as label. Hybridization was performed overnight at 65°C and the following series of washes done: 2X
SSPE/0.1 % SDS for five minutes at room temperature (RT); 2X SSPE/0.1 % SDS
for 10 minutes at RT; 1X SSPE/0.1% SDS~ for 15 minutes at 65°C; and O.1X
SSPE/0.1% SDS for 15 minutes at 65°C. Membranes were then exposed to autoradiographic film at -75°C with an intensifying screen.
Plugs in the vicinity of positive plaques were pulled, and each plug was placed into 1 ml of SM buffer to allow the phage particles to diffuse out.
Secondary and tertiary screens were performed using only single plaque lifts with the same protocol but scaled down for 100 mm plates. In further purifying the plaques, 500 particles were examined per plate in the secondary screen and 50 particles per plate in the tertiary screen.

The cDNA inserts of positive clones after the tertiary screen were subcloned into a pBS plasmid (ZAP construct) using an in vivo excision protocol supplied by the manufacturer (Stratagene). 11~I13K07 helper phage were utilized and transformed cells were grown overnight at 42°C on LB/Amp plates to eliminate the problem of helper phage co-infection. Inserts were sized using a restriction enzyme double digest using EcoR:I and XhoI.
DNA Sequence Analysis Preliminary DNA sequences were cabtained manually by the dideoxy chain termination method for the PCR product subclone and the HRADI cDNA
ZAP construct. This was performed with a TT sequencing kit containing T7 DNA
Polymerase (Pharmacia Biotech) according to the instructions of the manufacturer. The "read short" protocol was followed and [a 'SS]dATP was used as label. For the PCR product subclone, sequencing reactions utilized a T7 primer, while for the HRADI cDNA ZAP construct, an SK primer was used.
Samples from the sequencing reactions were resolved on a vertical 8%
acrylamide sequencing gel for two hours a1: 90 W. The gel was fixed in 10%
acetic acid/ 10% methanol for 20 minutes, blotted on 3 mm filter paper and dried on a vacuum gel dryer for two hours at 8(1°C. The gel was exposed to film (Kodak X-OMAT AR) overnight at RT arid the film developed on a Kodak M35A X-GMAT developer.
The confirmed HRADI cDNA ZAP construct in the correct orientation had both strands of the full insert sequenced by the automated sequencing facility at Queen's University (Kingston, Ontario, Canada) in the Department of Biochemistry. This facility utilizes the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli'Taq DNA Polymerase, FS. Samples were prepared in deionized distilled w2~ter (dHzO) with DNA (plasmid) concentrations of 1 ~,g/~l and synthetic oligonucleotides were supplied in concentrations of 1 pmol/ul. T3 and T7 primers were used in the first rounds of sequencing and synthetic oligos were made to sequences further downstream of these primers in order to obtain the full sequences of the inserts. The sequences of the synthetic oligos were as follows: Oligo E:

GTGTGTGCAAGCAAATGCTT (+ strand corresponding to the T3 primer) (SEQ ID N0:23); and Oligo F: CAAGGTGGGAACTTCCTGCA (- strand corresponding to the T7 primer) (SEQ ID N0:24).
S. pombe growth and manipulation S. pombe was cultured by standard tE;chniques (Leupold, U., Meth. Cell Physiol. 4:169-177 (1970)). To determine whether the isolated human cDNA
clone could rescue the UV sensitivity of an S. pombe rad 1-1 mutant, a radl -1 leu-1 strain, Sp263 (Davey , S., and Beach, D., Mol. Biol. Cell. 6:1411- 1421 ( 1995)), was transformed with the full length HRADl construct in the pART 1 expression vector (Bali, G. et al., Gene 123::131-6 (1993)) by the lithium acetate method (Okazaki, K., et al., Nucleic Acids I~'es. 18:6485-9 (1990)). These were plated on pombe minimal (PM) agar to select for transformants and grown at 30°C for three days. Cultures were then grown in PM broth to midlogarithmic ("midlog") phase at 25°C.
For radiation sensitivity analyses, cE;lls were plated in triplicate on PM
agar for each dose. Plates were either irradial:ed with a 254 nm UV light at a dose rate of 4 J/m2/sec, at doses of 0 to 100 J/m2, or were gamma irradiated in a Clinac 2100 C/D with a 6-MV beam, at a dose rate of 0.23 Gy/sec, at doses of 0 to 300 Gy. Plates were incubated at 30°C for seven days and the total number of yeast colonies on each plate were counted. 'The average of the three plates were normalized to the unirradiated control, and the graphed results were used as an indicator of viability by comparing them to the values of the wild type strain subjected to the same treatment and to the wlirradiated plates.
For viability in hydroxyurea (HLI), midlog phase cultures were treated with 10 or 12 mM HLI, and aliquots were taken at various time points. Aliquots were diluted appropriately, and plated on minimal selective media to maintain the HRADl expression plasmid. Plates were incubated for seven days at 30°C, and counted. Average surviving fractions were normalized to untreated samples harvested immediately prior to HTJ addition.

Yeast Two Hybrid Library Screen HRAD9 cDNA was subcloned from pBluescript into the Smal and Sall restriction sites of the GAL4 DNA binding .domain pGBT9 vector (Clontech).
pGBT9-HRAD9 was then transformed into the budding yeast strain HF7c (Feilotter et al., Nucl. Acids Res. 22:150:2-1503 (1994)) according to the manufacturer's instructions. Transformants were plated on synthetic dropout (SD) media minus tryptophan (6.7 g/L DII~ CO Yeast Nitrogen Base without Amino Acids, 2% glucose, 0.62 g/L Bio 1 X01 Complete Supplement Mixture minus histidine (-his), leucine (-leu), and tr,~ptophan (-trp), 20 mg/L
histidine, 100 mg/L leucine, and 20 g/L DIFCO Bacto-Agar), also referred to herein as "triple dropout medium". To ensure that the HRAD9 GAL4 DNA binding domain hybrid construct alone did not activate the HIS3 and/or lacZ reporter genes, colonies were streaked onto the S~D agar-trp-his and tested for ~i-galactosidase activity using a filter assay described in the Clontech Manual.
One HF7c colony harboring the pGBT9-HRAD9 vector was picked into 150 ml of SD-trp liquid media and grown to saturation :for two days at 30°C. The saturated culture was then diluted by adding 1 L of YTD (10 gA yeast extract, 20 g/1 tryptone, and 20 g/1 dextrose) and grown to an ODD of 0.5. These yeast were then transformed, as described by the manufacturer, with 0.5 mg of a directionally cloned HeLa cDNA library vn the pGAD-GH GAL4 activation domain vector (Clontech). The transformalits were plated on forty-four 15 cm plates containing SD agar-trp-leu-his and incubated at 30°C. To determine the efficiency of the library transformation, serial dilutions of a small aliquot of the transformed yeast were plated on SD agar-tr~~-leu. After 10 days, approximately 500 colonies grew larger than background on the triple dropout plates. These colonies were subcultured onto SD agar-trp-leu-his + S mM 3-aminotriazole (3-AT) and incubated at 30°C for 2 days, after which 15 positive clones were identified. Plasmid DNA was then prepared from 15 saturated liquid cultures essentially as described by the manufacturer (Clontech). XL1 -Blue competent bacteria were then transformed with this DNA and plated on LB agar containing ampicillin. Inserts in pGAD-GH were sequenced using fluorescently labeled SK
primer and an automated sequencer (AEd). DNA sequence analysis was performed using the BLAST algorithm (Altschul et al., J. Mol. Biol. 215:403-(1990)).
For analysis of individual interactions between HRAD9, HRAD1, and hHUS 1, HF7c were simultaneously cotransformed with pGBT9-hHUS 1 and pGAD-HRAD1, pGBT9-HRAD9and pGAI)hHUSl, and pGBT9-HRAD9 and pGAD-HRAD1, as described by the manufac;turer(Clontech). Cotransformants;
were plated on SD agar-trp-leu and incubated at 30°C for 2-3 days. As negative controls, pGBT9 fusion constructs were co-transformed with empty pGAD-GH
vector and pGAD fusion constructs were c;otransformed with empty pGBT9 vector. As a positive control, a p53-DNA binding domain fusion construct was co-transformed with a pSV40 T antigen-activation domain fusion construct. A
single isolated colony from each plate was streaked onto both SD agar-trp-leu and SD agar-txp-leu-his + SmM 3-AT and grown at 306C for 2-3 days.
HRAD9 Polyclonal Antibody Prepu~ration and Purification HRAD9 cDNA was PCR cloned into the SmaI and BamHI restriction sites of the pGEXI bacterial expression vector (Pharmacia). aHRAD9-Glutathione S-transferase (GST) fusion probein was then expressed in E coli and affinity purified on Glutathione Sepharose (Pharmacia) according to previously described methods (Frangioni et al., Anal. Biochem. 210:179-187 (1993)). a-HRAD9 polyclonal chicken antibodies were generated against this HRAD9 fusion protein (RCH antibodies).
10 mg of purified GST was batch adsorbed to 2 ml of glutathione sepharose for 2 hours at 4°C. Sepharose w~~s washed with 40 volumes of PBS.
2 ml of antibody supernatant was batch adsorbed with the GST bound glutathione sepharose overnight. Sepharose was subjected to centrifugation and the supernatant harvested.
pg of purified GST-HRAD9 protein was subjected to electrophoresis through a 10% acrylamide gel, then elc;ctro-blotted onto a nitrocellulose membrane. The protein band was visualized by Ponceau S staining and the band 30 excised and cut into small pieces with a scalpel. Membrane pieces were blocked overnight in 1 % casein in PBST (PBS + 0. 1 % Tween 20) at 4°C in a microfuge _77_ tube. The membrane was then washed 3 timc;s for S minutes each in PBST. I ml of pre-cleared antibody supernatant was added to the membrane pieces and rocked at 4 ° C for 4 hours. The supernatant was removed and the membrane was washed 2 times rapidly and once for 15 minutes with PBST. The tube was centrifuged briefly and all traces of the wash were removed. The antibody was eluted from membrane with 300 ~,1 of 0.2 M glycine pH 2.8. A second elution with 100 ~.1 of glycine was pooled with the first and the antibody supernatant was neutralized with 0.2 volumes of 1 M Tris pH 8Ø
Co-immunoprecipitation Experiments Co-immunoprecipitations utilized the myc and FLAG epitope tags, and for simplicity, proteins expressed with these tags are denoted by a subscripted m or f, respectively. HRAD1 cDNA was amplified by PCR and cloned into the XbaIlXhoI restriction sites ofthe mammalian. expression vector, pyDF31 (Gift of Dr. David LeBrun (Dept. of Pathology, Queen's University, Kingston, Ontario, Canada), in frame with one copy of the FLAG epitope. HRAD9 cDNA was PCR
cloned into the XbaIlXhoI restriction sites of the pCS2-MT, a mammalian expression vector with 6 copies of the myc; epitope (Rupp et al., Genes Dev.
8:1311-1323 (1994); Turner et al., Genes Dev. 8:1434-1447 (1994)). A hHUS 1 -myc fusion construct was generated by PCR amplifying hHUS 1 cDNA and cloning it into the pCS2-MT vector. The constructs used to express the negative controls FLAG epitope-tagged hepatic leukemia factor (HLFf) and FerONm.
Constructs were gifts of Dr. David LeBrun arid Dr. Peter Greer (Cancer Research Laboratories, Queen's University), respectively.
COS-1 cells that were approximatc;ly SO% confluent in 10 cm tissue culture plates were transiently co-transfecte;d with 24 ~g each of the indicated constructs. using lipofectin reagent (SIGM:A) according to the manufacturers instructions. Cells were then washed twice with 10 ml of sterile PBS, and 10 ml of complete DMEM was added (DMEM + 11J% fetal bovine serum). Transfected cells were cultured at 37 ° C in a 5 % COz al:mosphere for 48 hours.
Cells were lysed directly on the plate in mammalian cell lysis solution (50 mM Tris-Cl pH
8.0,150 mM NaCI, 0.5% NP40,1 mM Na3VIJ4,1 mM PMSF, 20 wg/ml aprotinin, _7$_ ~g/ml leupeptin). Lysates were passed through 18 and then 23 gauge syringes several times to shear genomic DNA, incubated on ice for 30 minutes, and centrifuged at 16,000 x g to remove any insoluble material. Each co-transfected cell lysate was split into two equal portions. 'Co one set, lysates were pre-cleared 5 with 3 5 ~,l of a-IgY agarose (Promega) on a :Nutator at 4 ° C for 45 minutes, and immunoprecipitated with polyclonal chicken anti-HRAD9 antibodies on a Nutator at 4°C for 1 hour. 'These immune complexes were collected on 35 ~,1 of a-IgY agarose (Promega) at 4 ° C for 1 hour. To the other set, lysates;
were pre--cleared with 10 wl protein G-sepharose (Pharlacia) and immunoprecipitated with 10 approximately 1 ~g of a-myc 9E10 mouse monoclonal antibody (Sigma, and gift of Dr. Peter Greer). These immune complexc;s were collected on 10 ~,1 of protein G-sepharose at 4°C for 1 hour. Both the a-myc and a-HRAD9 immunoprecipitated complexes were collected by centrifugation at 500 x g, washed four times with PBS, and incubated at 100°C for 5 minutes in 50 wl of SDS-PAGE sample buffer (NEB). Following centrifugation at 16,000 x g for 20 minutes, supernatants were electrophoresed through a single 6% acrylamide gel.
Protein was transferred to nitrocellulose (0.2 hum pore size, Xymotech) which was blocked in 5% MPBST (PBS + 5% non-fat milk powder + 0. 1 % Tween 20) at room temperature for 2 hours, and then probed with a-myc 9E 10 mouse monoclonal antibody. After extensive washing in PBST, HRP-conjugated anti-mouse secondary antibody was added and the membrane was incubated for 45 minutes at room temperature. Protean antigens were detected by chemiluminescence using the ECL detection system (Amersham), followed by exposure to X-ray film (Kodak). Three tines less a-myc immunoprecipitate sample (12 ~ul) was loaded onto the gel than ix-HRAD9 sample (4 ~1).
Similarly, the a-myc immunoprecipitate side of the blot was exposed to film for one fifth the time that the a-HRAD9 immunoprecipitate side.
For the HRAD 1 ~hHLlS 1 and HRAD~~/HRAD 1 co-immunoprecipitations, the same methods as described for the HRAL>9/hHUS 1 co-immunoprecipitations were used, with the following exceptions. .All lysates were pre-cleared with ~1 protein G-sepharose (Pharmacia) on a Niutator at 4°C for 45 minutes.
Either a-myc 9E10 monoclonal antibody or a-FLAG M2 monoclonal antibody (Sigma) was used for immunoprecipitation. Samples were size-fractionated on 10%
polyacrylamide gels. Immunoblotting was cauried out using a-myc 9E 10 mouse monoclonal or a-FLAG M2 monoclonal antibody, as indicated.
Calf Intestinal Phasphatase Treatments COS-1 cells were transfected with 24 pg of pCS2-MT-HRAD9 as described previously. Two days after the transfection, cells were harvested and immunoprecipitated with a-myc monoclonal antibody as before. After collecting the immune complexes on protein G-sepharose, beads were washed four times with PBS and resuspended in 200 pl of NEB buffer 3 (50 mM Tris-HCI, 10 mM
MgClz,100 mM NaCI, and 1 mM DTT) + 1 '% SDS. Protein was removed from the sepharose beads by heating at 100 ° C for 5 minutes followed by centrifugation at 16,000 x g. 20 pl of the supernatant was then treated with 30 units of calf intestinal alkaline phosphatase (Promega) in 1 x NEB buffer 3 in the presence or absence of 2 M sodium orthovanadate (Na3V04), for 30 minutes at 37°C.
To sufficiently dilute the SIDS in the sample, the total volume of the reactions was 200 pl. Both reactions, along with 20u1 of untreated immunoprecipitate, were made up to 1 ml with PBS, and re-immunoprecipitated with a-myc monoclonal antibody, electrophoresed through 6% acrylamide, and immunoblotted with a-myc monoclonal antibody essentially as above.
Endogenous HRAD9 protein was immunoprecipitated from approximately 9 x 106 HeLa cells with polyc.lonal chicken a-HRAD9 antibodies essentially as described above. The phosphatase procedure followed was identical to that for exogenous HRAD9m e~;cept samples were electrophoresed through 8% acrylamide and immunoprecipitated and immunoblotted with a-HRAD9 antibodies.
HRAD9 Immunofluorescence HaCaT or HeLa cells were seeded on coverslips for 1 hour (HeLa) or overnight (HaCaT) at 37°C in 5% CO2. Cellls were washed twice with PBS
and fixed with 10% paraformaldehyde for 10 minutes at room temperature. Fixed cells were washed twice more with PBS, cowered with methanol, and incubated at -20°C for 20 minutes. Cells were rinsed twice, and then washed for 30 minutes in PBST. PBST + 1 % normal goat serum (N~GS) was used to block cells at room temperature for 1 hour. Incubation in polyclonal a-HR.AD9 chicken antibodies in PBST + 1 % NGS for 1 hour at room temperature was followed by two rinses, and one 30 minute wash in PBST. Cells were then incubated in Alexa 488 goat anti-chicken secondary antibody (Molecular Probes) and diluted to 10 pg/ml in PBST + 1 % NGS for 1 hour at room temperature. After two rinses with PBST, and two 10 minute washes in PBS, cells were treated with 200 ug/ml RNase A
in 1 % PBS for 1 hour at 37°C. After two vrinses and two 5 minute washes in PBST, nuclei were stained with 2 pg/ml pro:pidium iodide in PBS for 5 minutes at room temperature. Cells were rinsed twice and washed once for 10 minutes with PBST. Coverslips were mounted on glass slides and visualized using a Meridian Insight Plus confocal microscope. Images were captured from a cooled Meridian video with a Matrox 1280 frame gzabber (Matrox Electronic Systems Ltd.) and pseudocolored and saved using MC;ID M4 software (Imaging Research Inc.).
Example 1: Isolation of the HRADI and MRADI Genes HRADI
A search of the dBEST database (National Center for Biotechnology, National Institutes of Health, Bethesda, Maryland) revealed an EST of interest (484 nucleotides) obtained from a normalized and directionally cloned human cDNA library. The complimentary strand of the EST appeared to encode a predicted polypeptide similar to a portion of the S. pombe radl + gene product.
This open reading frame predicted a polype:ptide that is 30% identical and 57%
similar over an 80 amino acid stretch, which represents approximately one quarter ofthe RADlp protein. It is aligned closer to the C-terminal portion of the S. pombe protein which is a moderately conserved region shared by the Schizosaccharomyces pombe radl+, Saccharomyces cerevisiae RAD17, and Ustilago maydis RECl gene products. The extent of homology in the region that the EST is aligned with S. pombe radl+ is comparable to that of radl+ and RADl7 (Lydall, D. and Weinert, T., Science 270:1488-91 (1995)). This same region contains 9 identical residues between RAD 1 p, RAD 17p, and RECIp, of which 7 are also present in the human EST. Based on the alignment and extent of sequence identity, this was evidence for the existence of a possible human orthologue of S. pombe radl+.
Because a positive orientation clone was not present in the dBEST library, we chose to search other cDNA libraries for the bona fide human radl homologue. A HaCaT (spontaneously transi:ormed human keratinocyte) cDNA
library in ~. ZAP II was amplified by PCR using oligonucleotides directed against the putative HRADI gene. PCR amplification using HaCaT cDNA resulted in PCR products generated from the expected primer pairs. The 399 by PCR product generated using oligonucleotides A and B (SI:Q ID NOs:17 and 18, respectively) was subcloned into pBS KS-. Sequencing of the subclone revealed an insert of identical sequence to that of the original EST, confirming that the sequence of interest was present in the HaCaT cDNA library.
The screen of 106 phage particles eventually yielded 4 positive clones after a tertiary screen. In vivo excision converted these ~, cDNA vectors into pBS
plasmids containing the cDNA insert (ZAf construct). Two of these, clones HRAD 1-7 and HRAD 1-8, were in the orientation expected for HRAD 1 sense expression. The other two, clones HRAI)1-9 and HRAD1-10, were in the opposite orientation. Clone HRAD 1-7 was slightly longer than clone HRAD 1-8, and so was chosen for further analysis.
MRADI
The full length HRAD 1-7 clone was used to probe a mouse CB7 erythroleukemia cDNA library by low stringency hybridization. Five positives were identified, four of which were the same length, and one was slightly shorter than the others. Clone MRAD1-2.1 was chosen for further analysis.
Complete sequencing of both strands of clone MRAD 1-2.1 identified a cDNA
that was 1380 by long with a 218 by 5' U'TR, an 840 by coding region and a 322 by 3' UTR (Figure 2; the additional 12 by shown in this figure are the 6 by EcoRI sites flanking the 5' and 3' ends of the MRAD1-2.1 cDNA). The 3' UTR contains a common variant of the consensus polyadenylation signal sequence (ATTAAA). However, no poly(A) tail is observed in this cDNA
isolate. The ORF of MRADl encodes a 280 amino acid polypeptide that is 90 ~
identical and 96 % similar to HRAD lp. Ami:no acid alignment (Figure 3) shows that the sequence similarity of HRADlp and MRADlp to the other members of the Radlp family extends over their entire lengths, suggesting that the isolated human and mouse cDNAs are full llength.
Example 2: Sequence Analysis of the H.RADl and MRADl Genes Full DNA sequences of both strands of the insert of clone HRAD1-7 showed that the cDNA was 1300 by long witlh a 214 by 5' untranslated region, an 846 by coding region and at least a 240 by 3' untranslated region (Figure 1 ).
The ORF of HRADI encodes a 282 amino acid pc~lypeptide. This is 40 as shorter than the S. pombe radl+ gene product. An aligxlment of HRADIp to Radlp was generated using the CLUSTAL W program (Thompson, J.D., et al., Nucleic Acids Res. 22:4673-4680 ( 1994)). Figure 3 shows that HRAD 1 p and the other members of the RAD 1 p family share protein sequence similarity over their entire lengths, suggesting that the isolated human cDNA is full length. The extent of the similarity between HRADIp (SEQ ID 110:2) and Radlp (SEQ ID N0:26) (27% amino acid identity, 53% similarity) is comparable to that between Radlp (SEQ ID N0:26) and RADl7p (SEQ ID NC1:27) (23% identity, SO% similarity) over their respective full lengths (Lydall, D. and Weinert, T., Science 270:1488-91 (1995)). As shown in Figure 4, the similarity between HRADIp (SEQ ID
N0:2) and the corresponding murine radl+ orthologue MRADlp (SEQ ID
N0:4), was significantly greater, exceeding about 90-95% amino acid identity and similarity.
Example 3: HRADl Partially Rescues the G2 DNA Damage Checkpoint Defects of radl:: ura4+ Yea;et The HRADI ORF was subcloned into the S. pombe expression vector pARTl under control of the strong, constitutive adhl+ promoter. Expression of HRADI in a radl:: ura4+ strain background increased the viability of these mutants following UV irradiation, to levels above that of the vector-transformed control (Figure SA). However, this increase in viability did not reach the level of rescue that was obtained by expression of the wild type radl+ gene (Figure SA).
Expression of HRADI also restored partial resistance to ionizing radiation (Figure SB).
In order to more rigorously examine; if HRADI rescues the checkpoint defects of radl mutants, HRADl was expressed in a radl-1 strain containing the temperature sensitive cdc25-22 allele. At the restrictive temperature of 36 ° C
these yeast arrest at the G2/M transition point:, due to their inability to activate the Cdc2 kinase. If cells blocked at the G2/M transition are irradiated just prior to being released to the permissive temperature of 25 ° C, checkpoint proficient cells will undergo a dose dependent delay in entry into mitosis. Checkpoint deficient cells will enter mitosis without a noticeable delay. As shown in Figure 6A, the checkpoint-deficient vector transformed controls entered a synchronous mitosis within 100 minutes of being irradiated, regardless of the dose received.
Checkpoint-proficient yeast expressing Rad lp underwent the characteristic dose dependent delay in entry into mitosis (Figure 6B). Yeast expressing HRADlp also underwent a dose dependent delay in entry into mitosis (Figure 6C). The dose dependence is not equal to that of cells. expressing Radlp; however, this is what one would expect for partial rescue.
Expression of HRADI was found to restore only limited resistance in rad l: : ura4+ yeast to the DNA synthesis inhi bitor hydroxyurea (HL)).
Expression of HRADI in Sp337 (Radl null strain) increased the viability of these yeast after exposure to HU. As shown in Figure 7, HRADI -expressing cells did not lose viability to as great a degree as vector-transformed control cells. Cells expressing wild type radl + remained viable to a much greater extent than either of the other samples.
Example 4: Generation of MRADI'' (l~Iul1 Mutant) Cell Lines The MRADI cDNA is used as a probe to obtain genomic clones using standard screening procedures (Sambrooh, J., et al., Molecular Cloning: A

Laboratory Manual, 2nd Ed., Cold Spring H~~rbor Laboratory Press, Cold Spring Harbor, NY (1989)). Preferably, a positive-negative selection scheme is used (Mansour, S. L., et al., Nuture 336:348-352 (1988)) to produce "knockout" or null mutant cell lines. As large a region as possible of the genomic locus containing coding exons is removed and the positive selection cassette (neo' gene under control of the phosphoglycerate kinase [PGK] promoter) inserted in its place. The PGK-neo cassette is flanked on both sides by 2-5 kb of homology to the target locus. Outside the region of homology the negative selection cassette (HSV thymidine kinase [TK] under control of the PGK promoter) is inserted.
Generation of targeted embryonic stem (ES) cells is performed essentially as described (Mortensen, R. M., Proc. Natl. Acad. Sci. USA 88:7036-7040 (1991)). Briefly, ES cells grown to 7'_.% confluence are harvested and trypsinized; 10' cells are suspended in 1 ml of 20 mM HEPES, 145 mM NaCI, and 0.1 mM 2-mercaptoethanol containing 1 pmol of the targeting construct.
Electroporation is performed in a Bio-Rad Gene Pulser at 450 V and 250 mF.
Cells are then plated at 5 x 106 cells per 1:i0-mm gelatinized Petri dish.
After 2 days, cells are selected with 6418 at 0.15 mg/ml and gancyclovir (GANC) at 2 mM. G418 selects for cells which have integrated the targeting construct.
GANC, which is converted to a cytotoxic compound by HSV TK, selects against 6418-resistant cells which arise by random integration of the targeting construct.
6418-resistant cells which result from integration due to homologous recombination at the target locus lose the PGK-HSV TK cassette. Cells are cultured for an additional 7-10 days with daily medium changes, after which surviving colonies are counted, isolated, an<t expanded. Single disruptions of the target locus are confirmed by genomic Southern blots using both locus-specific probes that recognize sequences located outside the targeting construct and the neo gene as a probe.
At this stage, transgenic cell lines and animals are prepared by any of several approaches. In a first approach, M~RADI+~- heterozygous ES cell clones are injected into mouse blastocysts in order to generate chimaeric mice. These chimaeric mice are then mated to produce ~fRADI-'' nullizygous embryos. From these embryos mouse embryonic fibroblast~; (MEFs) are produced using standard procedures (Deng, C., et al., Cell 82:675-684 (1995)). Day 11.5 to 16.5 post coitum embryos are dissociated, treated with DNase and trypsin, and plated in Dulbecco's modified Eagle's medium (DDrIEM) containing 15% fetal bovine serum (FBS).
In a second approach, provided the mice are viable, the mice are allowed to mature and primary tissue cultures are prepared from their mammary epithelium as previously described (Johnson, C. W., et al., CancerRes. 45:3774-3781 (1985)). Briefly, mammary tissue is removed, minced, and washed in phosphate-buffered saline (PBS). Tissue is dissociated by sequential digestion with collagenase III, trypsin, and DNase I in Ca2+/Mgz+-free PBS. Cells are washed with PBS and plated in DMEM containing 15% FBS.
A third option is the method of Mortensen et al. to generate homozygous null ES cells (Mortensen, R. M., et al., Mol. Cell. Biol. 12:2391-2395 (1992)).
Singly disrupted ES cells are expanded for 14-28 days, plated, cultured for 24 hours, and then selected at concentrations of 6418 up to 2.0 mg/ml. Doubly disrupted ES cell clones are confirmed by g~enomic Southern blotting. MRADI''' nullizygous ES cells are then allowed to dii:~erentiate in vitro, if desired.
Example 5: Generation of Human Cell Lines Deficient in HRADI
Expression To generate HRADl' human cell lir,~es, the full length HRADI cDNA is cloned in the reverse orientation in the mammalian expression vector pcDNA3 (Invitrogen). HaCaT cells (spontaneously transformed human keratinocytes) are transfected with this antisense HRADl expression construct by electroporation (Bio-Rad Gene Pulser, as above). Stable transfectants are selected and maintained in DMEM supplemented with 10% FBS and 6418 at 1 mg/ml. HRADI
expression levels are determined by northern blot analysis and immunoblot analysis using polyclonal antisera generated as described below in Example 7.
Vector-transfected cells may be used as controls for the experiments described below in Example 6.

Example 6: Analysis of RADl-deficient Cell Lines Once mammalian RADl -deficient cells and cell lines have been generated by the above-described approaches, they are then examined for their biological characteristics (e.g., cell cycle kinetics and checkpoint control). The techniques used to assess cell cycle kinetics and checkpoint characteristics are the same regardless of the cell type used in the experiments.
A. Cell Cycle Analysis Cell cycle analysis involves meas~uing the doubling time of RADI-deficient cell lines, compared to control cells (either normal MEFs or vector-transfected HaCaT cells). If a change in doubling time is observed, fluorescence-activated cell sorting (FACS) analysis (facili.ties available at Queen's University, Kingston, Ontario, Canada) is performed to detenmine which phase of the cell cycle is perturbed. A dual 5-bromo-2'-deoxy~uridine (BrdU)/propidium iodide (PI) FACS analysis allows resolution of the Gl, S, and G2+M phases of the cell cycle.
Cells are incubated for 1-4 hours at 37°C in the presence of BrdU, harvested, stained for BrdU incorporation with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibodies and for DNA content with PI, and subjected to FACS analysis as described (Davey, S., and Beach, D., Mol. Biol. Cell 6: 1411-1421 (1995)). Analysis of mitotic entry kinetics is performed with cells that are pulse-labelled with BrdU and followed through mitosis. BrdU is incorporated into cells which are in S phase at the time of labelling. Therefore, the time required for BrdU-labelled cells to complete mitosis represents the amount of time necessary for these cells to finish replication, transit G2, and complete mitosis. Completion of mitosis is monitored using FACS analysis, in which case the end of mitosis is evidenced by a return of BrdU-labelled cells to a G1 DNA
content. Alternatively, mitotic entry is measured by counting mitotic figures under a fluorescence microscope. This method uses cells that are pulse-labelled with BrdU and then grown in the presence of colcemid in order to prevent exit from mitosis.
B. Response to DNA Synthesis Inhibition andRadiation Treatment These experiments allow determinatiion ofthe cell cycling parameters and viability of RADI -deficient cells following .exposure to agents such as the DNA
synthesis inhibitor hydroxyurea (HU), UV radiation, and y radiation. Mitotic entry and cell cycle kinetics are determined as described above, and viability is assessed by plating efficiency.
HU inhibits ribonucleotide reductase, preventing dNTP synthesis and blocking replication (Elford, H. L. Biochem. Biophys. Res. Comm. 33:129-135 (1968)). Cells are labelled with BrdU, then treated with 0.2 mM HU and followed through mitosis using the methods described above. To assess viability, cells are exposed to HU for varying lengths of time (0-8 hours for yeast cells; 0-72 hours for human cells), then released from the HU replication block and plated.
Resulting colonies are fixed, stained, and counted. In response to HU, checkpoint-proficient control cells will delay mitosis until DNA replication is complete. If RAD 1-deficient cells are checkpoint-defective, as expected, they do not delay mitosis in response to HU, but rather undergo mitosis with kinetics similar to untreated cells.
Radiosensitivity of RAD1-deficient cells is determined by plating efficiency. y radiation is delivered using a. "'Cs source (available at Queen's University, Kingston, Ontario, Canada), and 254 nm UV light is delivered using a calibrated UV light box. Cells are irradiatead with 0-10 Gy of'y radiation or 0-20 J/mz of UV light and plated. Colonies are fi:ced, stained, and counted. Cell cycle kinetics are followed at varying time point:. after irradiation. The dose used, as well as the timing, depend on the results obtained above. In response to radiation, checkpoint-proficient cells delay mitosis, allowing time for DNA repair to occur.
RADl -deficient cells, presuming they are ch<;ckpoint-defective, enter mitosis with the same kinetics as untreated cells.
C. Determination of Genomic Instability As described above, checkpoint dE;fects are associated with genomic instability. Amplification of the CAD gene. is used as an indicator of genomic instability in RADI -deficient cells. Amplil:ication of the CAD gene results in resistance to the drug N-(phosphonacetyl)-L,-aspartate, or PALA (Otto, E., et al., _$8-J. Biol. Chem. 264:3390-3396 (1989); Tlsty, T. D., et al. Proc. Natl. Acad.
Sci.
USA 86:9441-9445 (1989)). PALA resistance occurs very rarely in normal cells, including human keratinocytes (Wright, J. ~~., et al., Proc. Natl. Acad. Sci.
USA
87:1791-1795 ( 1990)), making the frequency of PALA-resistant colony formation a good indicator of genomic instability (Otto, E., et al., J. Biol. Chem.
264:3390-3396 (1989); Tlsty, T. D., et al. Proc. ~fatl. Acad. Sci. USA 86:9441-9445 (1989)). The frequency of PALA-resistant colony formation in RAD1-deficient cells is determined, as compared to control cells. The assay is performed as described (Yin, Y., et al., Cell 70:937-948 (:1992); Livingstone, L. R., et al., Cell 70:923-935 (1992)), including selection in 55-88 mM PALA for 1-4 months, followed by fixation, staining, and scoring :for colony formation.
Example 7: Generation of anti-HRADIIp Polyclonal Antibodies The full length HRADI cDNA i s cloned in the vector pGEX2T
(Pharmacia, Piscataway, New Jersey) in frame with the glutathione-S-transferase (GST) moiety. Recombinant GST-HRADIp is purified from bacterial lysates by affinity chromatography, using Glutathione-Sepharose (Pharmacia) as previously described (Smith, D. B., and Johnson, K. S., Gene 67:31-40 (1988)). Multiple injections of 50-100 mg ofrecombinant GST-HRADIp are used to immunize a rabbit. The animal is boosted and bled three 'times, and polyclonal anti-HR.AI) 1 p antibodies are affinity purified from the antisera using recombinant GST-HRAD lp.
Example 8: Interaction of HRADlp with BRCAlp, ATRp or ATMp To determine if BRCAIp, ATRp, and ATMp proteins interact with HRAD 1 p, two sets of experiments using anti-HR.AD 1 p antibodies are conducted.
In a first experiment, anti-HRADIp antibodies are used in co immunoprecipitation experiments. In an alternative approach, recombinant GST-HRADIp protein is used in pull-down experiments to determine if HRADlp interacts with BRCAlp, ATRp, or ATMp proteins.
A. Co-immuhoprecipitations WI38 normal human fibroblasts or HaCaT cells are lysed in SO mM Tris-HCl (pH 8.0),150 mM NaCI, 0.5% NP-40 containing phosphatase inhibitors (50 mM NaF, 1 mM Na3V04) and protease inhibitors (aprotinin [20 mg/ml], leupeptin [ 10 mg/ml], and phenylmethylsulphonylfluoride [ 100 mg/ml]), at 4 °C.
S Lysates are pre-cleared with protein A-Sepharose (Pharmacia) and then precipitated with antibodies against either HF;AD 1 p, BRCAIp, ATRp, or ATMp, at 4 ° C. Immunoprecipitates are collected on protein A-Sepharose beads, separated by SDS-PAGE, and transferred to nitrocellulose membranes.
Immunoblot analysis is performed as follows. The membranes are blocked with low-fat milk powder, incubated with primary antibody (either anti-HRADlp,-BRCAIp, -ATRp, or -ATMp), and then incubated with species-specific, horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive proteins are detected by ECL (Amersham).
B. Pull down Experiments Pull-down experiments can be performed to confirm those of the co-immunoprecipitation experiments described .above. Recombinant GST-HRAD lp is bound to Glutathione-Sepharose beads. Lysates (prepared as described above) of cells expressing BRCAIp, ATRp, and ATMp are incubated with these beads.
Bead-bound protein complexes are washed, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Imlnunoblot analysis is then performed as described above.
Example 9: Co-localization of HRADlp with BRCAIp, ATRp or ATMp WI38 cells are used for the immunolocalization experiments because this is one of the cell lines in which co-localization of BRCAlp and HRADS lp has been shown (Scully, R., et al., Cell 88:265-275 (1997)). Immunostaining of adherent cells grown on cover slips is performed as described (Eckner, R., et al., Genes Dev. 8:869-884 (1994)). Cells are fixed in 3% paraformaldehyde/2%
sucrose in PBS for 10 min at room temperature, washed twice with PBS, and then permeabilized in ice-cold Triton X-100 buffer (50 mM NaCI, 3 mM MgCl2, 200 mM sucrose, 10 mM HEPES [pH 7.~1], 0.5% Triton X-100) for 5 min.

Primary antibody incubations are for 30 min at 37 °C, followed by three washes for 5 min each with PBS. Species-specific, iluorochrome-conjugated secondary antibodies are incubated in a similar fashion. Immunofluorescence is recorded using a Zeiss confocal microscope (available at Queen's University, Cancer Research Laboratories, Kingston, Ontario, (Janada).
General Discussion for Examples 1-9 We have identified novel human and mouse genes that are structural homologues of the fission yeast radl+ checkpoint control gene. The sequence similarity extends over the entire coding regions, indicating that the isolated cDNAs are fixll length. Particularly high levels of conservation were seen in the putative exonuclease domains, as well as in the leucine-rich region, which have been previously defined (Onel, K., et al., Genetics 143:165-174 (1996)). The extent o f amino acid conservation between BRAD 1 p and rad 1 p, 27% identity and 53% similarity, is comparable to that observed between Radlp and RADl7p (23% identity, 50% similarity), which have been shown by independent means to be involved in checkpoint control in fission and budding yeast, respectively (Lydall, D., and Weinert, T., Science 27(7:1488-1491 (1995)). In different regions, HRADlp (SEQ ID N0:2) and MRADlp (SEQ ID N0:4) appear more like each of Radlp (SEQ ID N0:26), RADl7p (SEQ ID N0:27), and RECIp (SEQ ID N0:25).
While it has previously been clearly demonstrated that REClp (SEQ ID
N0:25) is a 3'-5' exonuclease, it has also beE,n demonstrated that this fiznction is not required for checkpoint control by this. protein (Onel, K., et al., Genetics 143:165-174 (1996)). The sequence similaril:y between HRAD lp, MRAD lp, and other members of the family over the exo II and exo III domains is high, but less high in the exo I domain.
In the present studies, we were able: to show that HRADl can partially rescue radiation sensitivity in radl: : ura4~~ mutant yeast, but fails to rescue hydroxyurea sensitivity. This partial complementation indicates that HRADI is a likely human homologue of fission yeast radl+. Cross-species complementation by checkpoint genes has been demonstrated in other cases, but full complementation of all the defects of an:y particular mutant has not been observed. For example, the human homologue of S. pombe rad9+ restores resistance to treatment with hydroxyurea to the rad9 null mutant, but fails to rescue LTV sensitivity (Lieberman, H.B., eat al., Proc. Natl. Acad. Sci. USA
93:13 890-13 895 ( 1996)). FRPI lATR has al.;o been shown to rescue some of the checkpoint defects in MECl mutant S. cerevisiae, but not in rad3- S. pombe (Bentley, N.J., et al., EMBO J. 15:6641-66~~ 1 (1996)).
In mammalian cells, the G1 checkpoint is regulated in part by the p53 and ATM genes, and defects in these genes have been associated with a variety of human cancers (Livingstone, L. R., et al., Cell 70:923-935 ( 1992); Yin, Y., et al., Cell 70:937-948 (1992); Malkin, D., et al., Science 250:1233-1238 (1990);
Srivastava, S., et al., Nature 348:747-749 (1.990); Swift, M.., et al., N.
Engl. J.
Med. 325:1831-1836 (1991); Savitsky, K., et al., Science 268:1749-1753 (1995);
Dulic, V., et al., Cell 76: 1013-1023 (1994); Kastan, M.B., et al., Cancer Res.
51:6304-6311 (1991); Kuerbitz, S.J., et al., l°roc. Natl. Acad. Sci.
USA 89:7491-7495 (1992); O'Connor, P.M., et al., CancerRes. 53:4776-4780 (1993); Kastan, M.B., et al., Cell 71:587-597 (1992); Swift, M., et al., New Engl. J. Med 216:1289-1294 (1987)). By contrast, very little is known about the molecular control of the G2 checkpoint in mammalian cells. Like yeast, mammalian cells will respond to DNA damage or incompletc;ly replicated DNA by arresting the cell cycle in G2, prior to entry into mitosis. The presence of such a G2 checkpoint has been shown to correlate with viability after exposure to radiation (Cheong, N., et al., Mutat. Res. 274:111-122 (1992); Knox, S.J., et al., Radiat.
Res.135:24-31 (1993); McKenna, W.G., et al., Radiat. Res.125:283-287 (1991);
Nagasawa, H., et al., Int. J. Radiat. Biol. 66::573-379 (1994); Su, L.N., and Little, J.B., Radiat. Res. 133:73-79 (1993)).
As a result of the present invention, there are now three candidates for human G2 checkpoint control genes: h~RADl , HRAD9, and FRPI lATR, homologues of the radl+, rad9+, and rad3'+ genes of S. pombe, respectively.
HRAD1, HRAD9 and ATR have been shown to rescue some of the defects in checkpoint-deficient fission and budding; yeast. Interestingly, BRCAIp colocalizes with the repair protein RADS l p, and both are found in regions of meiotic chromosomes similar to where FRF' lp/ATRp is located (Keegan, K.S., et al., Genes Dev. 10:2423-2437 (1996); Scully, R., et al., Cell 88:265-275 (1997)). Genetic evidence from yeast indicates that radl+, rad3+ and rad9+ are part of the same G2 checkpoint control pathway, and may form a physical complex. This suggests that HRADIp, as the homologue of Radlp, may be part of a multisubunit complex that includes other checkpoint proteins, including HRAD9p, FRPlp/ATRp, RADSlp, and BR.CA1.
It has been shown that caffeine treatment partially restores sensitivity to radiation in cell lines which have lost G1 checkpoint control through the loss of p53 (Russell, K.J., et al., Cancer Res. 55:1639-1642 (1995); Yao, S.-L., et al., Nature Med. 2:1140-1143 ( 1996)). Presumalbly, the loss of the ability to undergo apoptosis in response to radiation in p53 mutant cells leads to radiation resistance. Caffeine is presumed to eliminate the G2 checkpoint in these cells, leading to radiation-induced death by premature mitosis, typical of checkpoint-defective cells. Directly targeting HRADI or HRAD 1 p could be an efficient way of targeting human G2 checkpoint control. If elimination of G2 checkpoint function would restore sensitivity to radiation or chemotherapeutic drugs to cells which have lost G1 checkpoint function (e.g.,. p53-'- cells), there will be therapeutic benefits to inhibiting HRADl or other G2 checkpoint control genes and protein functions, in conjunction with radio- or chemo- therapies.
Introduction for Examples 10-12 As noted above, the eukaryotic cell cycle consists of a number of tightly regulated events whose precise order ensures that the important tasks of DNA
replication and cell division occur with high fidelity. Cells maintain the order of these events by making later events dependent on the successful completion of earlier events. This dependancy is enfon;ed by cellular mechanisms called checkpoints (Weinert, T.A., and Hartwell, L.H., Science 241:317-22 (1988);
Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol. 10:6554-64 (1990)). The DNA damage (G2) and DNA replication (S-phase) checkpoints arrest eukaryotic cells at the G2/M transition in the presence of damaged or incompletely replicated DNA, respectively (Weinert, ~C.A., and Harlwell, L.H., Science ' CA 02268457 1999-04-19 241:317-22 ( 1988); Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol.
10:6554-64 ( 1990); Epoch et al. , Cell 60:665-673 ( 1990); al-Khodairy et al., EMBO
J.
11:1343-1350 (1992); Epoch, T., et al., Genes Dev. 6:2035-46 (1992); Rowley, R., et al., EMBOJ. 11:1335-42 (1992); al-Klaodairy et al., Mol. Biol. Cell 5:147-160 ( 1994)). This arrest provides time for tree cell to repair damage or complete replication prior to entry into mitosis.
Evidence according to the best current model indicates that in the fission yeast Schizosaccharomyces pombe, the ultimate target of the G2 checkpoint signals is the tyrosine 15 residue of the cyclin dependent kinase Cdc2 (Epoch et al., Cell 60:665-673 ( 1990); O'Connell et al., EMBO.L 16:545-554 ( 1997);
Rhind et al., Genes Dev. 11:504-S11 (1997)). '.Phosphorylation of this residue is regulated primarily by the Cdc25 phosphatase and the Weel protein kinase, and the activity of these enzymes is regulated in turn by the kinases Chkl and Cdsl, respectively (Walworth et al., Nature 363:368-371 (1993); Furnari et al., Science 277:1495-1497 (1997)). Chkl is only required for the DNA damage checkpoint (Walworth et al., Nature 3b3:368-371 (199?) and functions by phosphorylating and inhibiting Cdc25, thereby preventing Cdc2 dephosphorylation and mitotic entry (Fumad et al. 1997). When the S-phase checkpoint is triggered, activation of Cdsl results in activating phosphorylation of Weel, which then results in inhibitory phosphorylation of Cdc2 (Boddy et al., Science 280:909-912 (1998)).
While the mechanistic detail involved in the; G2 checkpoints upstream of these proteins is unclear, it is known that a group of six proteins in fission yeast are required for both G2 and S-phase checkpoint control. These proteins are Radl, Rad3, Rad9, Rad 17, Rad26, and Hus 1, and are collectively termed the checkpoint rad proteins (al-Khodairy and Carr, EMBO,1. 11:1343-1350 (1992); Epoch, T., et al., Genes Dev. 6:2035-46 (1992); Rowlt;y, R., et al.) EMBO J. 11:1335-42 (1992); al-Khodairy et al., Mol. Biol. Cell .S: 147-160 (1994)). Evidence that these genes are all critical components of both the damage and replication checkpoints is based on observations that t:he checkpoint rad mutants, unlike wild-type cells, do not block mitotic entry in response to DNA-damaging agents or transient inhibition of DNA synthesis (al-Khodairy and Carr, EMBO J.
11:1343-1350 (1992); Epoch, T., et al., Genes Dev. 6:2035-46 (1992); Rowley, R., et al., EMBO J. 11:1335-42 (1992); al-Kt~odairy et al., Mol. Biol. Cell 5:147-160 (1994)). The checkpoint rods are placed upstream of the Cdc2 regulators in the emerging checkpoint signal transduction pathway because the checkpoint-induced phosphorylation of the Chkl and Cdsl kinases are dependent on the presence of all of the checkpoint rod proteins (Walworth et al., Science 271:353-356 (1996); Lindsay et al., Genes Deu 12:382-395 (1998)). More recently, it was shown that Rad 1 and Hus 1 form a stable complex that is dependant on Rad9, suggesting that these three proteins may exist in a three way.-complex in fission yeast (Kostrub et al., EMBO J. 17:2055-2066 (1998)).
Many of the genes involved in the G2 checkpoint pathways are conserved between humans and yeast. By the present invention and other work, human homologs of all of the fission yeast checkpoint rods, except Rad26, have been identified, suggesting that the fission yeast G2 checkpoint signaling mechanism may be similar to that of humans (Cimprich et al., Proc. Natl. Acad. Sci. USA
93:2850-2855 (1993); Lieberman, H.B., et al., Proc. Natl. Acad. Sci. USA
93:13890-13895 (1996); Kostrub et al.) EMBOJ. 17:2055-2066 (1998); Parker et al., J. Biol. Chem. 273:18332-18339 (1f98); Parker et al., J. Biol. Chem.
273:18340-18346 (1998); Udell et al., Nucl. Acids Res. 26:3971-3978 (1998)).
In accordance with this hypothesis, Chk2, the :human equivalent of S. pombe Cdsl protein kinase, has recently been shown to block Cdc2-regulated mitotic entry in response to G2 checkpoint activation, by phosphorylating Cdc25C (Matsuoka et al., Science 282:1893-1897 (1998)). Furthermore, this response is dependent on ATM, a human homolog of fission yeast Rad3 (Savitsky, K., et al., Science 268:1749-53 (1995); Savitsky et al., Hum. Mol. Gen. 4:2025-2032 (1995)).
Therefore, the human equivalents of the checkpoint rods appear to be functioning upstream of the Cdc2 regulatory machinery, .as they do in fission yeast.
In the following Examples, we identify further conservation between the fission yeast and human G2 checkpoints by demonstrating that human homologs of S. pombe checkpoint rods, HRAD 1, HRAfr~9, and hHUS 1, physically interact with one another in vivo. We have also previously shown that endogenous HR.AD9 is phosphorylated and that it localizes primarily to the nucleus in unperturbed HeLa and HaCaT cells.

Example 10: Interaction Between hHUSl and HFtADl An interaction between Hus 1 and Ra.dl has been previously described in S. pombe (Kostrub et al., EMBO J. 17:2055-2066 (1998)). Therefore, we investigated whether a similar interaction existed between hHUS 1 and HRAD 1.
We co-transformed pGBT9-hHUSI and pGAD-HRAD1 into HF7c and looked for activation of the HIS3 reporter gene b;y subculturing co-transformants on triple dropout media (Figure 8a). The same positive and negative controls were used as before. While neither fusion plasmid on its own was sufficient for growth in the absence of histidine, co-transformation of pGBT9-hHUS l and pGAD-HRAD 1 resulted in viable HIS+ co-transformants. These results suggested that a specific interaction existed between hHUSI and HRAD1.
To confirm this interaction, exogenously expressed HRAD1 and hHUS 1 were co-immunoprecipitated in COS-1 cells (Figure 8b) using FLAG epitope-tagged HRAD 1 and myc epitope-tagged hHUS 1. HLFf and FerONm were included as negative controls to ensure the specificity of the interaction.
Cells were co-transfected as indicated with HF;AD 1,lhHUS 1 m, HLF,JhHUS 1 m, or HRAD I,JFerONm. The cells were harvested 48 hours after transfection, lysed, and immunoprecipitated with either a-FLAG M2 monoclonal anitbody or a-myc 9E10 monoclonal antibody. Two aliquots lFrom each sample were subjected to electrophoresis through two identical polyacrylamide gels, one of which was used for an a-FLAG western blot and the other for an a-myc western blot. Although exogenous HRAD 1 f protein levels were approximately equivalent in both pyDF31-HRAD1 transfections (Figure 8b, lanes 1 and 3), HF,AD1 f immunoprecipitated only with hHUS lm (Fii;ure 8b, lane 1) and not with FerONm (Figure 8b, lane 3). Similarly, hHUSIm immunoprecipitated with HRADIf (Figure 8b, lane 1 ), but not with HLFf (Figure 8b, lane 2). Together, these results indicate the existence of a specific physical interaction between hHUS 1 and HRAD 1.
Example 11: Interaction Between HRAlD9 and HF~AD1 Having observed the two interactions described in Example 10, we next explored the association status of HRAD~> and HR.AD1. Using the pGBT9-HRAD9 and pGAD-HRAD 1 GAL4 fusion constructs, we repeated the yeast two-hybrid experiment described above (Figure 9a). Despite growth of the p53/pSV40 T-Ag positive control (Figure 9a, lower left quadrant), co-expression of HRAD9 and HRAD 1 filsions failed to assc;mble a filnctional GAL4, and hence did not produce viable yeast in the absence of histidine (Figure 9a, lower right quadrant). Therefore, while the yeast two-hybrid system demonstrated interactions between hHUS 1 and HRAD9, and hHUS 1 and HRAD 1, it showed no interaction between HR.AD9 and HRAD 1.
To confirm this result, we examined the ability of HRAD9 and HRAD 1 to co-immunoprecipitate in COS-1 cells (Figure 9b). HRAD9m and HItAD 1 f were exogenously expressed either together, or separately with HLFf or Fer~Nm as described in Example 10. Neither HRAID 1 nor HRAD9 protein expression levels varied significantly between different transfections. Contrary to the yeast two-hyrbrid data, HRAD9m but not Fer~N"" immunoprecipitated with HRAD 1"
and HRAD 1 ~, but not HLF~, immunoprecipitated with HRAD9m. Therefore, while HItADl and WAD9 show no interaction in the two-hybrid system, they do specifically co-immunoprecipitate with each other when exogenously expressed in COS-1 cells.
Example 12: Association of H1RAD1 with Phosphorylated HltAD9 In immunoprecipitations performed using antibodies directed against either native or epitope tagged HRAD9 (see Example 11, Figure 9), we noted multiple discrete bands in western analyses. We have gone on to demonstrate that these bands are the result of phosphorylation, using both epitope tagged exogenously expressed I~AD9 in COS-1 cells, as well as using endogenous HRAD9 in HeLa cells (Figure 10). COS-~ 1 cells were transfected with our HRAD9",y~ expressing construct, and harvested 48 hours later. Alternatively, HeLa cells were simply harvested at SO% confluency. The cells were lysed and immunoprecipitated with 9E 10 monoclonal antibody directed against the myc epitope, or with a-HRAD9 antibodies, respectively. Immunoprecipitates were either untreated, or treated with calf intestinal phosphatase (CIP) in the presence or absence of the phosphatase inhibitor sodium orthovanadate. Samples were then subjected to SDS polyacrylamide ;gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunolblotted with a-myc or a-H1RAD9 antibodies. Figure 10a, lane 1, shows the multiple banding pattern of HRAD9my~
in immunoblots, similar to that seen in Figure 9. Treatment with C1P caused the slower migrating bands to disappear, leaving only the fastest form. This effect was eliminated by the presence of vanadate, confirming that the slower migrating bands are the result of multiple phorphoryla~tion states of HRAD9mY~. In HeLa cells, only a single slower migrating band was observed (Figure 1 Ob, lane 1 ). The HRAD9 can be converted to the faster migxating, de-phosphorylated form by treament with CIP, and this reaction was also found to be sensitive to sodium orthovanadate (Figure l Ob, lanes 2 and 3), indicating that endogenous HRAD9 is phosphorylated in HeLa cells.
We have also noted that the most heavily phosphorylated forms of HRAD9 are preferentially bound by HRAD1 (Figure lOc). An immunoprecipitation of HRAD9 followed by an a-HRAD9 western shows approximately equal amounts of each phosphorylated form of H1ZAD9 are present in cells, while if the same sample is immunoprecipitated with a-Flag (HRAD1), followed by a HRAD9 western;, the most heavily phosphorylated forms of H1RAD9 are selectively, thoul;h not exclusively, precipitated.
Confirmatory dephosphorylation experiments on HR.AD9 in HRAD 1 immunoprecipitations are shown on the right hand side of Figure 1 Oc. This is the first indication that phosphorylation of HltAD9 influences complex formation with other members of the G2 checkpoint protein family.
General Discussion for Examples 10-12 We have demonstrated three interactions between three human checkpoint rad proteins, HRAD1, »tAD9, and hHUS 1. In all cases, these interactions were substantiated using both the yeast tyro-hybrid system and by co-immunoprecipitation, except for HRAD 1 and H1RAD9 which did not interact in the yeast two-hybrid, but did co-immunoprecipitate when exogenously expressed in COS-1 cells. The original observation that led to this work was that H1RAD9 interacted with hHUS 1 in a two-hybrid screen. Nine of 15 interactions isolated in the screen that used HRAD9 as bait were 11HUS 1. It is interesting to note that HRAD 1 was not among the remaining isolates; however, the observation that HRAD 1 and HRAD9 show no interaction in the two-hybrid confirms previous results (Parker et al., J. Biol. Chem. 273:18332-18339 (1998); Parker et al., J.
Biol. Chem. 273:18340-18346 (1998)). It now appears that this interaction may be dependant on factors that are absent in budding yeast, since HRAD 1 and HRAD9 specifically co-irnmunoprecipitate in COS-1 cells. Such factors may include a budding yeast equivalent of hHLJS 1, the existence of which seems unlikely considering that no homologs have been identified based on sequence.
Alternatively, the N-terminal GAL4 domain of the fusion proteins may result in a conformational change that prevents association of these two proteins. This hypothesis is supported by our observation 'that reversing the orientation of the HRAD9/ hHUS 1 and HRAD 1 / hHUS 1 C?AL4 fusions, abolishes the HIS3 reporter gene activation (data not shown). Furthermore, an N-terminal myc tagged version of fission yeast Hus 1 has bec,n shown to function as a dominant negative allele (Kostrub et al. , Mol. Gen. Genet. 254:389-399 ( 1997)).
Future use of dominant negative fusions involving human proteins could prove invaluable in uncovering the mechanistic details involved in checkpoint signaling.
It has been shown in fission yeast that Husl and Radl interact, and that this interaction is dependent on the presence of Rad9, as interaction does not occur in a rad9 null background (Kostrub et czl., EMBO J.17:2055-2066 ( 1998)).
Our data presented here offer strong evidence that such a complex also exists in humans, although it may be assembled differently. While we have examined pairwise interactions between the three human checkpoint proteins in the present examples, the simplest explanation of the present data and the yeast data together is that a three way complex exists between HRAD1, IiRAD9, and hHUSl. We cannot rule out the possibility that the ob;cerved HRAD1-hHUSI interaction described here are bridged by DDC1, the S. cerevisiae homolog of HRAD9 (Longhese et al., EMBO J. 16:5216-5226 (1!97); Paclotti et al. 1998), or by and endogenous monkey homolog of HRAD9 iin COS-1 cells: such evidence will ultimately have to be achieved using HRAI)9 null cell lines. Further, with the highly similar phenotypes observed in all ~of the fission yeast checkpoint rad .

mutants, and considering recent data delr~onstrating an interaction between HRAD1 and HRAD17 (Parker et al., J. Biol. Chem. 273:18332-18339 (1998);
Parker et al., J. Biol. Chem. 273:18340-18346 (1998)), and that ATR, a human homolog of fission yeast Rad3, exists predominantly as part of a high molecular weight complex (Wright etal., Proc. Natl. Acad. Sci. USA 95:7445-7450 (1998)), the potential for a multi-protein complex involving all of the checkpoint rad proteins must not be overlooked.
We have also shown here that both exogenous and endogenous HRAD9 is phosphorylated at multiple sites. Considtering that S. cerevisiae DDC 1 and S. pombe Hus 1 both appear to be phosphorylated in response to DNA damage (Kostrub et al., Mol. Gen. Genet. 254:389-399 (1997); Paciottl et al. 1998), phosphorylation is an integral component of checkpoint signaling. To determine if checkpoint activation affects HRAD9 phosphorylation, we investigated whether y-radiation or hydroxyurea could induce a change in the migration pattern of endogenous HRAD9 on a western blot. Neither a 4 Gy dose of gamma radiation, nor incubation in 0.1 mM hydrox~rurea for up to 24 hours affected the migration of endogenous HRAD9 from HaC'aT cells, though HRAD9 is already highly phosphorylated in these cells. We cannot rule out the possibility that ongoing replication or the presence of endogenous DNA damage may be inducing HRAD9 phosphorylation in the absence of exogenous signals. It is worth noting that phosphorylation is not an absolute requirement for association of HRAD9 and HRAD1, as a HR.AD1 immunoprecipitation will co-immunoprecipitate all forms of HRAD9, although the present data demonstrated that the most highly phosphorylated forms were preferentially co-precipitated.
Finally, we have investigated the sub-cellular localization ofHR.AD9, and we have shown that HRAD9 is a nuclear protein. This observation was not a foregone conclusion, as the start of the checkpoint signal transduction pathway is nuclear (DNA damage), while the end is cytoplasmic(the cell cycle machinery).
Unlike HRAD1, which has been shown to be present mainly in a diffuse pattern in the nucleus (Freire et al., Genes Dev. x'2:2560-2573 (1998)), the staining pattern of HRAD9 within the nucleus shows discrete areas of intense staining.

As alluded to above, one reason for the current intense interest in cell cycle checkpoint control is the association of defects in checkpoint control with human cancers. Genomic instability is a common feature accompanying checkpoint loss, regardless of which checkpoint is compromised, and whether or S not the cell is subjected to exogenous stresses (Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol. 10:6554-64 (1990); Livings;tone et al., Cell 70:923-935 (1992);
Yin, Y., et al., Cell 70:937-48 (1992)). A great deal of evidence now links genomic instability with the mufti-step oril;in of human cancer (Loeb, Cancer Res. 51:3075-3079 (1991); Hartwell, Cell 71:543-546 (1992); Meyn, CancerRes.
55:5991-6001 (1995); Smith et al., Curr. Opin. Oncol. 7:69-75 (1995); Thrash-Bingham et al., Cancer Res. 55:6189-6195 (1995); Tlsty et al., Mutat. Res.
337:1-7 (1995); Loeb et al., Mutat. Res. 350:279-286 (1996); Perucho, Biol.
Chem. 377:675-684 (1996)). The number ofcheckpoint control genes which act as tumor suppressors under normal circumstances is growing, and currently includes p53 (Malkin, D., et al., Science 2~~0:1233-1238 (1990); Kastan et al., Cell 71:587-597 (1992); Kuerbitz et al., Proc. Natl. Acad. Sci. USA 89:7491-7495 (1992)), ATM (Savitsky, K., et al., Science 268:1749-53 (1995); Savitsky et al., Hum. Mol. Gen. 4:2025-2032 (1995); Xu et al., Genes Dev. 10:2401-2410 (1996)), and hBUBl (Cahill et al., Nature 392:300-303 (1998)). While none of the checkpoint rad proteins has yet been shown to act per se as a tumor suppressor, several have been mapped to regions associated with loss of heterozygosity in human tumors, which is :indicative of the presence of tumor suppressing genes Lieberman, H.B., et al., Proc. Natl. Acad. Sci. USA 93:13890-13895 (1996); Parker et al., J. Biol. Chem. 273:18332-18339 (1998); Parker et al., J. Biol. Chem. 273:18340-18346 (1998)). Also, genomic instability has been associated with G2 checkpoint deficiency in budding yeast rad9 mutants (Weinert, T.A., and Hartwell, L.H., Mol. Cell. Biol. 10:6554-64 (1990)).
Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the practice of the invention within a wide and equivalent -1~1-range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be: incorporated by reference.

Claims (79)

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:1;
(b) a nucleotide sequence encoding the HRAD1p polypeptide having the complete amino acid sequence set forth in SEQ ID NO:2;
(c) a nucleotide sequence encoding the HRAD1p polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 011905 arid which was deposited on ~ at ~ as deposit number ~;
(d) the nucleotide sequence of an HRAD1-encoding polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:1; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d).

2. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:3;
(b) a nucleotide sequence encoding the MRAD1p polypeptide having the complete amino acid sequence set forth in SEQ ID NO:4;
(c) a nucleotide sequence encoding the MRAD1 polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on ~ at ~ as deposit number ~ ;
(d) the nucleotide sequence of an MRAD1-encoding polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:3; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d).

3. The nucleic acid molecule of claim 1 or claim 2, wherein said polynucleotide has a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to said reference sequence.

4. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence set forth in SEQ ID NO:1.

5. The nucleic acid molecule of claim 1 wherein said polynucleotide has a nucleotide sequence encoding the HRAD1p polypeptide having the complete amino acid sequence set forth in SEQ ID NO:2.

6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on ~ at ~ as deposit number ~.

7. The nucleic acid molecule of claim 1 wherein said polynucleotide has the nucleotide sequence of an HRAD1-encoding polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:1.

8. The nucleic acid molecule of claim 2 wherein said polynucleotide has the complete nucleotide sequence set forth in SEQ ID NO:3.

9. The nucleic acid molecule of claim 2 wherein said polynucleotide has a nucleotide sequence encoding the MRAD1p polypeptide having the complete amino acid sequence set forth in SEQ ID NO:4.

10. The nucleic acid molecule of claim 2 wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on ~ at ~ as deposit number ~.

11. The nucleic acid molecule of claim 2 wherein said polynucleotide has the nucleotide sequence of an MRAD1-encoding polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO:3.

12. An isolated nucleic acid molecule comprising a polynucleotide encoding an epitope-bearing portion of an HRAD1p polypeptide, wherein said epitope-bearing portion is selected from the group consisting of a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID NO:2; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about in SEQ ID NO:2; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in SEQ ID NO:2;
a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in SEQ ID NO:2; and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID NO:2.

13. An isolated nucleic acid molecule comprising a polynucleotide encoding an epitope-bearing portion of a MRAD1p polypeptide, wherein said epitope-bearing portion is selected from the group consisting of: a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID NO:4; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about in SEQ ID NO:4; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 112 to about 128 in SEQ ID NO:4;
a polypeptide having an amino acid sequence; consisting essentially of amino acid residues from about 159 to about 177 in SEQ ID NO:4; and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID No:4.

14. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

15. A recombinant vector comprising the isolated nucleic acid molecule of claim 2.

16. The vector of claim 14 or claim 15, wherein said vector is an expression vector.

17. A recombinant host cell comprising the nucleic acid molecule of claim 1 or the recombinant vector of claim 14.

18. A recombinant host cell comprising the nucleic acid molecule of claim 2 or the recombinant vector of claim 15.

19. A method for producing an isolated HRAD1p polypeptide, comprising culturing the recombinant host cell of claim 17 under conditions such that said polypeptide is expressed, and isolating said polypeptide.

20. A method for producing an isolated MRAD1p polypeptide, comprising culturing the recombinant host cell of claim 18 under conditions such that said polypeptide is expressed, and isolating said polypeptide.

21. An isolated HRAD1p polypeptide produced according to the method of claim 19.

22. An isolated MRAD1p polypeptide produced according to the method of claim 20.

23. An isolated HRAD1p polypeptide having an amino acid sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence; as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the HRAD1p polypeptide as set forth in SEQ ID NO:2; and (c) the complete amino acid sequence of the HRAD1p polypeptide encoded by the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on ~ at ~ as deposit number ~.

24. An isolated MRAD1p polypeptide having an amino acid sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:3;
(b) the complete amino acid sequence of the MRAD1p polypeptide as set forth in SEQ ID NO:4; and (c) the complete amino acid sequence of the MRAD1p polypeptide encoded by the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on ~ at ~ as deposit number ~.

25. The polypeptide of claim 23 or claim 24, wherein said polypeptide has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to said reference sequence.

26. An isolated polypeptide comprising an epitope-bearing portion of an HRAD1p polypeptide, wherein said epitope-bearing portion is selected from the group consisting of a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID NO:2;
a polypeptide having an amino acid sequence; consisting essentially of amino acid residues from about 89 to about 97 in SEQ ID NO:2; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about to about 128 in SEQ ID NO:2; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in SEQ
ID NO:2; and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID NO:2.

27. An isolated polypeptide comprising an epitope-bearing portion of an MRAD1p polypeptide, wherein said epitope-bearing portion is selected from the group consisting of a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 77 to about 86 in SEQ ID NO:4;
a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 89 to about 97 in SEQ ID NO:4; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about to about 128 in SEQ ID NO:4; a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 159 to about 177 in SEQ
ID NO:4; and a polypeptide having an amino acid sequence consisting essentially of amino acid residues from about 227 to about 257 in SEQ ID NO:4.

28. A method of producing an isolated HRAD1p-specific antibody comprising immunizing an animal with the isolated HRAD1p polypeptide of any one of claims 21, 23 or 26, and isolating an HRAD1p-specific antibody from said animal.

29. A method of producing an isolated MRAD1p-specific antibody comprising immunizing an animal with the isolated MRAD1p polypeptide of any one of claims 22, 24 or 27, and isolating an MRAD1p-specific antibody from said animal.

30. An isolated HRAD1p-specific antibody produced according to the method of claim 28.

31. An isolated MRAD1p-specific, antibody produced according to the method of claim 29.

32. The isolated antibody of claim 30 or claim 31, wherein said antibody is a polyclonal antibody.

33. The isolated antibody of claim 30 or claim 31, wherein said antibody is a monoclonal antibody.

34. The isolated antibody of claim 30 or claim 31, wherein said antibody is detectably labeled.

35. The isolated antibody of claim 30 or claim 31, wherein said antibody is immobilized on a solid support.

36. An antisense oligonucleotide which is complementary to an HRAD1 mRNA sequence corresponding to all or a portion of an HRAD1 nucleic acid molecule having a nucleotide sequence, as set forth in SEQ ID NO:1.

37. An antisense oligonucleotide which is complementary to an MRAD1 mRNA sequence corresponding to all or a portion of an MRAD1 nucleic acid molecule having a nucleotide sequence; as set forth in SEQ ID NO:3.

38. The antisense oligonucleotide of claim 36 or claim 37, wherein said oligonucleotide is a 15-mer to a 40-mer.

39. The antisense oligonucleotide of claim 36 or claim 37, wherein said oligonucleotide is a DNA molecule or a derivative thereof.

40. The antisense oligonucleotide of claim 36 or claim 37, wherein said oligonucleotide is a DNA phosphorothioate molecule or a derivative thereof.

41. A pharmaceutical composition comprising the antisense oligonucleotide of claim 36 or claim 37 and a pharmaceutically acceptable carrier or excipient therefor.

42. A ribozyme comprising a target sequence which is complementary to an HRAD1 mRNA sequence corresponding to all or a portion of an HRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID
NO:1.

43. A ribozyme comprising a target sequence which is complementary to an MRAD1 mRNA sequence corresponding to all or a portion of an MRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID
NO:3.

44. A pharmaceutical composition comprising the ribozyme of claim 42 or claim 43 and a pharmaceutically acceptable carrier or excipient therefor.

45. A transgenic animal comprising the isolated nucleic acid molecule of claim 1 or claim 2.

46. The transgenic animal of claim 45, wherein said animal is a transgenic mouse.

47. A method for determining the sensitivity of a first animal to a DNA-damaging agent, comprising:
(a) obtaining a first biological sample from said first animal and a second biological sample from a second animal of known sensitivity to the DNA-damaging agent;
(b) determining the level of expression of a mammalian RAD1 gene in the first and second biological samples; and (c) comparing the level of mammalian RAD1 gene expression in the first biological sample to the level of mammalian RAD1 gene expression in the second biological sample, wherein a lower level of mammalian RAD1 gene expression in the first biological sample relative to that of the second biological sample indicates a higher level of sensitivity to a DNA-damaging agent in the first animal relative to that of the second animal.

48. A method of diagnosing a disorder in a first animal suffering therefrom or predisposed thereto, wherein said disorder is characterized by increased sensitivity of the first animal to a DNA-damaging agent, comprising:
(a) obtaining a first biological sample from said first animal and a second biological sample from a second animal of known sensitivity to the DNA-damaging agent;
(b) determining the level of expression of a mammalian RAD1 gene in the first and second biological samples; and (c) comparing the level of mammalian RAD1 gene expression in the first biological sample to the level of mammalian RAD1 gene expression in the second biological sample.

49. The method of claim 47 or claim 48, wherein said DNA-damaging agent is radiation or a chemical.

50. The method of claim 47 or claim 48, wherein said mammalian RAD1 gene is an HRAD1 or an MRAD1 gene.

51. The method of claim 50, wherein said determination step (b) is accomplished by a technique that measures the amount of HRAD1 or MRAD1 mRNA present in said first and second biological samples.

52. The method of claim 51, wherein said technique is northern blotting.

53. The method of claim 50, wherein said determination step (b) is accomplished by a technique that measures the amount of HRAD1 or MRAD1 polypeptide present in said first and second biological samples.

54. The method of claim 53, wherein said technique is an immunological technique.

55. The method of claim 54, wherein said immunological technique comprises contacting said first and second biological samples with one or more antibodies that bind specifically to one or more HRAD1p or MRAD1p polypeptides present in said first and second biological samples.

56. A method of treating or preventing a disorder in a first animal suffering therefrom or predisposed thereto, comprising:
(a) obtaining a first biological sample from said first animal and a second biological sample from a second animal of known sensitivity to a DNA-damaging agent;
(b) determining the level of mammalian RAD1 gene expression in the first biological sample relative to that of the; second biological sample; and (c) treating said first animal, in which the the level of mammalian RAD1 gene expression in the first biological sample is different from that of the second biological sample, by a technique that cures, delays or prevents the development of, or induces remission of, said disorder.

57. The method of claim 56, wherein the level of mammalian RAD1 gene expression in the first biological sample is lower than that in the second biological sample.

58. The method of claim 56, wherein the level of mammalian RAD1 gene expression in the first biological sample is higher than that in the second biological sample.

59. The method of claim 56, wherein said mammalian RAD1 gene is an HRAD1 gene or an MRAD1 gene.

60. The method of claim 56, wherein said technique that cures, delays or prevents the development of, or induces remission of, said disorder, is selected from the group consisting of:
(a) chemotherapy;
(b) radiation therapy;
(c) surgery; and (d) a combination of two or more of the techniques in (a), (b) and (c).

61. The method of claim 59, wherein said technique that cures, delays or prevents the development of, or induces remission of, said disorder comprises introducing into said first animal a composition comprising one or more antisense oligonucleotides which are complementary to an HRAD1 mRNA sequence corresponding to all or a portion of an HRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1, or which are complementary to an MRAD1 mRNA sequence corresponding to all or a portion of an MRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID
NO:3.

62. The method of claim 59, wherein said technique that cures, delays or prevents the development of, or induces remission of, said disorder comprises introducing into said first animal a composition comprising one or more ribozymes comprising a target sequence which is complementary to an HRAD1 mRNA sequence corresponding to all or a portion of an HRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1, or which is complementary to an MRAD1 mRNA sequence corresponding to all or a portion of an MRAD1 nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:3.

63. The method of claim 61 or 62, wherein the introduction of said composition into said first animal induces a decrease in the expression of mammalian RAD1 in the animal.

64. The method of claim 61 or 62, further comprising at least one additional technique selected from the group consisting of chemotherapy and radiation therapy.

65. The method of claim 59, wherein said technique that cures, delays or prevents the development of, or induces remission of, said disorder comprises introducing into said first animal a composition comprising one or more isolated nucleic acid molecules having a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:1;
(b) a nucleotide sequence encoding the HRAD1p polypeptide having the complete amino acid sequence sea forth in SEQ ID NO:2;
(c) a nucleotide sequence encoding the HRAD1p polypeptide having the complete amino acid sequence encoded by the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on ___ at ___ as deposit number ____;
(d) the nucleotide sequence of an HRAD1-encoding polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having the nucleotide sequence as set forth in SEQ 1D NO:1; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) and (d).

66. The method of claim 65, wherein the introduction of said composition into said first animal induces an increase in the expression of HRAD1 in the animal.

67. The method of any one of claims 61, 62 or 65, wherein said composition further comprises a pharmaceutically acceptable carrier or excipient.

68. The method of any one of claims 61, 62 or 65, wherein said composition further comprises a vector or a virion.

69. The method of claim 68, wherein said vector or virion is derived from a retrovirus, an adenovirus or an adeno-associated virus.

70. The method of claim 48 or claim 56, wherein said disorder is a cancer.

71. The method of any one of claims 47, 48 or 56, wherein said first animal and said second animal are each a mammal.

72. The method of claim 71, wherein said mammal is a human.

73. A method of isolating a cell cycle checkpoint control polypeptide, comprising:
(a) providing a sample containing a cell cycle checkpoint control polypeptide to be isolated;
(b) contacting said sample with one or more isolated HRAD1p polypeptides under conditions favoring the binding of said cell cycle checkpoint control polypeptide to said isolated HRAD1p polypeptide; and (c) releasing said cell cycle checkpoint control polypeptide from said HRAD1p polypeptide, thereby isolating said cell cycle checkpoint control polypeptide.

74. A method of isolating a cell cycle checkpoint control polypeptide, comprising:
(a) providing a sample containing a cell cycle checkpoint control polypeptide to be isolated;
(b) contacting said sample with one or more isolated MRAD1p polypeptides under conditions favoring the binding of said cell cycle checkpoint control polypeptide to said isolated MRAD1p polypeptide; and (c) releasing said cell cycle checkpoint control polypeptide from said MRAD1p polypeptide, thereby isolating said cell cycle checkpoint control polypeptide.

75. The method of claim 73, wherein said isolated HRAD1p polypeptide has an amino acid sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:1;
(b) the complete amino acid sequence of the HRAD1p polypeptide as set forth in SEQ ID NO:2; and (c) the complete amino acid sequence of the HRAD1p polypeptide encoded by the cDNA clone having GenBank Accession Number AF 011905 and which was deposited on _____ at as deposit number ____.

76. The method of claim 74, wherein said isolated MRAD1p polypeptide has an amino acid sequence at least 65% identical to a reference sequence selected from the group consisting of:
(a) the amino acid sequence encoded by an isolated nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO:3;
(b) the complete amino acid sequence of the MRAD1p polypeptide as set forth in SEQ ID NO:4; and (c) the complete amino acid sequence of the MRAD1p polypeptide encoded by the cDNA clone having GenBank Accession Number AF 038841 and which was deposited on ____ at ____ as deposit number __.

77. The method of claim 73, wherein said HRAD1p polypeptide is immobilized on a solid support.

78. The method of claim 74, wherein said MRAD1p polypeptide is immobilized on a solid support.

79. An isolated cell cycle checkpoint control polypeptide produced according to the method of claim 73 or claim 74.