MXPA98001699A - Control point genes of the celu cycle - Google Patents

Abstract

The present invention relates to a class of checkpoint genes and their polypeptide products that control progression through the cell cycle in eukaryotic cells. Particularly, this invention relates to the Schizosaccharomyces pombe rad3 gene, its human homologue (ATR) and its encoded proteins. The invention further relates to test methods for selecting compounds that modulate the activity of the polypeptide products of these checkpoint genes and to the use of the selected compounds in an anti-cancer therapy.

Description

CELL CYCLE CONTROL POINT GENES The present invention relates to a class of checkpoint genes that control progression in the cell cycle in eukaryotic cells. BACKGROUND OF THE INVENTION The control of the cell cycle is fundamental for the growth and maintenance of eukaryotic organisms, from yeasts to mammals. Eukaryotic cells have developed control pathways, called "control points" that ensure that the individual steps of the cell cycle are completed before the next step occurs. In response to DNA damage, the survival of cells is iased both by direct DNA re-pairing mechanisms and by the delay in cell cycle progression. Depending on the position of the cell within the cycle at the time of irradiation, damage to DNA in mammalian cells can prevent (a) the passage of Gl to an S phase, (b) the progression in S phase or (c) the passage of G2 in myth is. It is agreed that these control points avoid negative events, such as the duplication of damaged DNA and the segregation of fragmented chromosomes during mitosis (Hartmell and Astan, 1994). The radi gene from Schi zosaccharamyces pombe is required for checkpoints that respond to DNA damage and replication blocks. Rad3 is a member of the kinase subclass called 1 ipidquinases which possess regions having sequence homology with the lipidkine domain of the pllO subunit of f asfatidi 1 ino-ol-3-kinase (PI-3 kinase). This subclass also includes the defective ATM protein in patients with ataxia-telangiectasia. Cells from patients with ataxia telangiectasia (AT cells) have lost the delay to the S phase after irradiation and are said to have a radioresistant DNA synthesis (Painter and Yourtg, 1989). AT cells irradiated in S phase accumulate in G2 with lethal damage, probably as a consequence of the attempt to duplicate the damaged DNA. AT cells irradiated during G2 have a different phenotype: they do not suspend mitosis after DNA damage, and progress through mitosis with damaged DNA (Beamish and Lavin, 1994). The mutation in the A-T locus, to which the gene of ATM has been mapped, resulting in a disorder of several control points required for an appropriate response to ionizing radiation. Other members of this subclass of lipidkinase include: Tellp (Greenwell et al., 1985). gene involved in the maintenance of an adequate length of tissue in Saccharomyces cersvisiae; Esrlpí Me ip and the product of the control point gene mei-41 from Dr sophila elanogaster ÍHari et al. nineteen ninety five). Presents ion of the invention. We have analyzed the rad3 gene of S.po be and found that it has a full length amino acid sequence of 2386 amino acids, not the 1070 amino acids described by Seaton et al. 1992. We determined that it is the direct homologue of the Esrlp gene of S. cerevisiae and that it shares the same globular structure as the ATM gene. The C-terminal region of the rad3 protein contains a lipidkinase domain that is required for the function of Rad3. We show that Rad3 can autoasaciarse. We also identified a protein kinase activity associated with Rad3. In addition, we found a human homologue of rad3. This gene, which we have called ATR (related to ataxia and rad), has a homologue that is slightly higher with rad3 than with the ATM gene. The sequence of ATP cDNA. human is presented in sequence ID No.l. The ORF amino acid sequence of nucleotides 80 and 8011 is presented in sequence ID No. 2. The DNA sequence of the open reading frame (0RF> of rad3 is illustrated in sequence ID No.3. the 2386 amino acids of the gene { nu leotids 585 to 7742 of Sequence ID No. 3) is illustrated in sequence ID No. 4. Accordingly, in a first aspect, the invention provides the ATP protein. of sequence ID No. 2 and homologs thereof, polypeptide fragments thereof, as well as antibodies capable of binding the ATP protein or polypeptide fragments thereof. The ATR proteins, homologs and fragments thereof are referred to below as polypeptides of the invention.

In another aspect, the present invention provides a polynucleotide in substantially isolated form capable of selectively hybridizing with sequence ID No. 1 or with the complement thereof (ie, opposite strand). Polynucleotides encoding polypeptides of the invention are also provided. Such polynucleotides will be known as polynucleotides of the invention. The polynucleotides of the invention include the Ha sequence ID DNA. 1 and fragments thereof capable of selectively hybridizing with this gene. In a further aspect, the invention offers recombinant vectors carrying a polynucleotide of the invention, including expression vectors, and methods for culturing these vectors in a suitable host cell, for example, under conditions in which the expression of a protein occurs. or polypeptide encoded by a sequence of the invention. In a further aspect, the invention provides sets of elements that include polynucleotides, psypeptides or antibodies of the invention and methods for using these sets of elements for the diagnosis of the presence or absence of ATR and its homologs, or variants of the same, including negative ATP mutants.

The invention further provides assay methods for screening candidate substances for use as compounds to inhibit or activate ATR activity, or the activity of mutated forms of ATR that are deficient in checkpoint activity. The invention also provides test methods for screening candidate substances for use as compounds to inhibit interactions in ATR and other compounds that interact with ATR, including ATR itself. In a related aspect, the invention also provides a polynucleotide sequence of sequence ID No. 3 in substantially isolated form, and sequence ID protein No. 4 in substantially isolated form, and novel fragments and variants thereof. DETAILED DESCRIPTION OF THE INVENTION A. Palukins The polynucleotides of the invention may comprise DNA or RNA. They can also be polynucleotides that include synthetic or modified nucleotides within them. Several different types of modification to oligonucleotides are known in the art. These types include methyl phosphonate and phosphorotide routes, addition of acridine chains or pol 1 isi a and the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it is understood that the polysides described herein can be modified by any other method available in the art. Such modifications can be carried out for the purpose of increasing the activity in vivo or the life of the polynucleotides of the invention. The polynucleotides of the invention that can hybridize selectively with the DNA of sequence ID No. 1 will have a homology of generally at least 70%, preferably at least 80 or 90% and with a greater degree of preference at least 95% with the corresponding DNA of sequence ID No. 1 in a region of at least 20, preferably at least 25 or 30, for example 40, 60 or 100 or more contiguous nucleotides. It will be understood that those skilled in the art can, using routine techniques, perform nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of a particular host organism wherein the polypeptides of the invention must be expressed. Any combination of the above-mentioned degrees of homology and minimum sizes can be used to define pol i. The invention is based on the invention, with preference being given to the most stringent combinations (ie higher homology in longer lengths). Accordingly, for example a polynucleotide having a homology of at least 80% on 25, preferably 30 nucleotides forms an aspect of the invention, as well as a polynucleotide having a homology of at least 90% on 40 nucleotides. The polynucleotides of the invention can be used to produce a primer, for example, a polymerase chain reaction primer, a primer for an alternative amplification reaction, a probe for example labeled with a developing marker by conventional means using radioactive or non-radioactive labels, or the polynucleotides can be cloned into vectors Such primers, probes and other fragments will have a length of at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides, and are also included within the term polynucleotides of the invention as used herein. Polynucleotides such as for example DNA polynucleotides and primers according to the present invention can be produced recombinantly, synthetically, or in any way available to those skilled in the art. They can also be cloned by standard techniques.

In general, the primers will be produced by synthetic means, including a gradual fabrication of the desired nucleic acid sequence, one nucleotide at a time. Techniques for achieving this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will include making a pair of primers (e.g., about 15-30 nucleotides) in a region of the ATR gene to be cloned, putting the primers in contact with mRNA or cDNA obtained from a human cell (e.g. dividing cell such as peripheral blood leukocyte), carry out a polymerase chain reaction under conditions that cause amplification of the desired region, isolating the amplified fragment (for example, by purification of the reaction mixture on a gel of agarose) and recovering the amplified DNA. The primers can be designed to contain suitable restriction enzyme recognition sites in such a manner that the amplified DNA can be cloned into a suitable cloning vector. Such techniques may be employed to obtain all or parts of the ATR sequence described herein. Genomic clones that contain the ATP gene. and their promoter regions and introns can also be obtained analogously, starting with the genomic DNA of a human cell, for example, a hepatic cell. Although, in general, the techniques mentioned herein are well known in the art, reference can be made in particular to Sambrook et al. (Molecular Cloning: A Labaratory Manual, 1989). Polynucleotides that do not have 100% homology for the sequences of the present invention but fall within the scope of this invention can be obtained in various ways. Other human allelic variants of the ATR sequence described herein can be obtained for example by the formation of probes from libraries of genomic DNA made from several individuals, for example individuals from different populations. In addition, ATR homologs can be obtained from other animals, particularly mammals (eg, mice, rabbits or rabbits), more particularly primates, and such homologs and fragments thereof may generally hybridize selectively with sequence ID No. 1. Such sequences can be obtained by forming probes from cDNA libraries made from dividing cells or tissues or genomic DNA libraries from other animal species, and forming probes with these libraries with probes comprising all or part of Sequence ID No. 1 ba or medium or highly stringent conditions (eg 0.03M sodium chloride and 0.03M sodium time at a temperature of about 50 ° C to about 60 ° C).

Allelic variants and homologs of species can also be obtained by employing degenerate polymerase chain reaction using primers designed to target sequences within the variants and homologs encoding conserved amino acid sequences. The conserved sequences can be predicted from the alignment of the amino acid sequence of ATR with that of rad3. The primers will contain one or several degenerate positions and will be used under stringent conditions lower than those employed for the cloning sequences with primers of unique sequences against known sequences. Alternatively, such polynucleotides will be obtained by site-directed mutagenesis of the ATR sequences or allelic variants thereof. This may be useful when, for example, silent codon changes are required to optimize codon preferences for a particular host cell in which polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Additional changes may be desirable to represent particular coding changes found in ATR that cause mutant ATR genes that have lost the checkpoint function.

Probes based on such changes can be used as diagnostic probes to detect such ATR mutants. The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement. The polynucleotides or primers of the present invention can carry a revealing label. Suitable labels include radioisotopes such as 32P or 35S, enzymatic labels, or other co-protein markers or for example biotin. Such labels can be added to the polynucleotides or primers of the invention and can be detected using techniques known per se.

Polynucleotides or primers of the invention or fragments thereof labeled or unlabeled may be employed by a person skilled in the art in nucleic acid-based assays to detect or sequence ATR in the human or animal body. Such tests for detecting generally comprise contacting a human or animal body sample containing DNA or RNA with a probe comprising an oligonucleotide or primer of the invention under hybridization conditions and detecting any duplex formed between the probe and nuc lei co acid in the sample. Detection can be achieved using techniques such as polymerase chain reaction or by immobilizing the probe on a solid support, removing the nucleic acid in the sample that is not hybridized with the probe, and then detecting the nucleic acid that was hybridized with the probe. Alternatively, the nucleic acid of the sample can be immobilized on a solid support, and the amount of probe attached to such support can be detected. Various suitable test methods of this or other formats can be found, for example, in WO 89/03891 and WD90 / 13667. Tests for the ATR sequence include contacting a human or animal body sample containing the DNA or the target RNA with a probe comprising a polynucleotide or primer of the invention under hybridization conditions and determining the sequence, for example by means of the Sanger dideoxy chain termination method (see Sambrook et al.). Such a method generally comprises elongation, in the presence of suitable reagents, of the primer by synthesis of a strand complementary to the DNA or target RNA and selectively terminating the elongation reaction in one or several residues of A, C, G or T / U; allowing the elongation and strand termination reaction to occur, separating the elongated products according to size to terminate the sequence of the nucleotides in which the selective termination occurred. Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides of dATP, dCTP, dGTP and dTTP, a regulator and ATP. Indenotides are used for selective termination. Tests for detection or sequencing of ATR in the human or animal body can be used to determine ATR sequences within cells in individuals who have or are suspected of having an altered ATR gene sequence, for example within cancer cells including leukemia cells and solid tumors such as ma ma a, ovaries, lungs, colon, pancreas, testes, livers, brain, muscle and bone. In addition, the discovery of ATR will allow to study the function of this gene in hereditary diseases, analogous to the ATR gene. In general, this will include the establishment of ATR status (for example using sequence analysis in polymerase chain reaction) in cells derived from patients with diseases that may be related to damage to replicating cells, for example, familial predisposition to cancer , chromosome rupture or phenotype of inescapability or phenotype of sensitivity to reparo-damage. The probes of the invention can be conveniently prepared in the form of a set of test elements in a suitable container. In such sets, the probes can be joined on a solid support where the format of the test for which the set of elements is designed requires such a connection. The set of elements may also contain suitable reagents for the treatment of the sample to be tested, hybridizing the probe with nucleic acid in a sample, control reagents, instructions, etc. The present invention also provides polynucleotides encoding the polypeptides of the invention described below. Because such polynucleotides will be useful as sequences for the recombinant production of polypeptides of the invention, it is not necessary that they can be selectively hybridized with the sequences of sequence ID No. 1 even though this is generally desirable. Otherwise, such polynucleotides can be labeled, used and prepared in the manner described above if desired. The polypeptides of the invention are described below. Particularly preferred polynucleotides of the invention are derivatives of the lipidkinase domain of ATR, its allelic variants and species homologs. The lipidkinase domain is represented by nucleotides 7054 through 8011 of sequence ID No.l. Polynucleotides of the invention which comprise this domain are preferred. The term "lipidkinase domain" refers to a domain that has a homology to other known 1-pidquases, particularly the 110-subunit of PI-3 kinase, in accordance with that determined by the sequence alignments. Other preferred polynucleotide pols of the invention are those comprising nucleotides encoding amino acids 181 to 302 of sequence ID No. 2 (nucleotides 620 to 985 of sequence ID Na. 1), which are considered to be a leucine closure region, A putative protein-protein interaction site, and amino acids 1358 to 1366 (nucleotides 4151 to 4177), which are also conserved. In a further aspect, polynucleotides of the present invention include those of sequence ID No. 3 and fragments thereof capable of hybridizing selectively with this sequence other than the fragments of nucleotides 2,462 to 6599 wherein the following changes were made: waste 2499, 2501, 2507 and 2509? insertion of C between 5918/5919. Particularly preferred fragments include those comprising residues 6826 to 7334 (the 1 ipidq? Inase domain) and the leucine binding regions 1476 to 1625 and 2310 to 2357. Addition- ally, the fragment comprising the conserved region 3891 is preferred. at 3917. Such fragments of palpeptides can be made and used in accordance with what has been described above. B. Polypeptides The polypeptides of the invention include polypeptides in substantially isolated form comprising the sequence presented in sequence ID No. 2. The polypeptides further include variants of such sequences, including naturally occurring allelic variants and variants. synthetics subetanc ially homologous to said polypeptides. In this context, a substantial homology is considered as a consequence having at least 70%, preferably 80% or 90% amino acid homology (identity) over 30 amino acids with sequence sequence ID No. 2, except in relation to with the lipidkinase domain and a C-terminal portion (residues 2326 to 2644) where a substantial homology is considered to be co or a homology of at least 80%, preferably 90% (identity) in 50 amino acids. Polypeptides other than those encoding ATR homologs are also included from other species including animals such as for example mammals (e.g., mice, rats or rabbits), especially primates, and variants thereof in accordance with the above defined. The polypeptides of the present invention also include fragments of the above-mentioned full-length polypeptides and variants thereof, including fragments of the sequence presented in sequence ID No. 2. Preferred fragments include those encompassing an epitope, specifically an epitope. Suitable fragments will have at least a size of about 5, for example 10, 12, 15 or 20 amino acids. Fragments of ATR protein polypeptides and allelic variants and of species thereof may contain one or more (eg, 2, 3, 5, or 10) their, deletions or. insertions, including conserved substitutions. The conserved substitutions can be elaborated according to the following table that indicates the conservative substitutions, where amino acids of the same block in the second column and preferably in the same row in the third column can be substituted between e11o: ALIPHATIC Non polar Polar GAPILV - without change CSTMN g Polar - changed DER AROMATIC HFY OTHERS NQDE Variants of the polypeptides of the invention can also comprise polypeptides where one or more of the specified amino acids fes naturally encoded) is removed or replaced or where one or more non-specific amino acids are replaced ( 1) if loss of kinase activity specific to the polypeptides of the invention; or (2) with the inactivation of the specific kinase activity of the polypeptides of the invention? or (3) with the inability to interact with members or regulators of the vis. of cell cycle control point. The epitopes can be determined either by techniques such as for example peptide explanatory techniques in accordance with that described by Geysen et al. Mol. Immupol., 23; 709-715 (1986). The polypeptides of the present invention may be in a substantially isolated form. It will be understood that the polypeptide can be mixed with carriers or diluents that do not interfere with the intended purpose of the polypeptide and that are considered to be substantially isolated. A polypeptide of the invention can also be in a substantially purified form, in which case, it will generally comprise the polypeptide in a preparation in which more than 90%, for example 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention. Polypeptides of the invention can be modified for example by the addition of histidine residues to aid in their purification or by the addition of a signal sequence to promote their secretion to par i r d a c 1 u1.

A polypeptide of the present invention may be labeled with a revealing label. The revealing label can be any suitable marker that allows detection of the polypeptide. Suitable labels include radioisotopes, for example 1251, enzymes, antibodies, polynucleotides and linkers, for example biotin. The labeled polypeptides of the invention can also be used in diagnostic methods such as for example immunoassays in order to determine the amount of a polypeptide of the invention in a sample. Marketed polypeptides or polypeptides of the present invention can also be employed in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols. A polypeptide or labeled polypeptide of the present invention or fragment thereof can also be fixed on a solid phase, for example the surface of an immunoassay well or a rod. Such labeled and / or immobilized polypeptides can be formed into sets of elements in a suitable container together with suitable reagents, controls, instructions and sires. Such polypeptides and sets of elements can be employed in methods of detecting antibodies to the ATR protein or its allelic or species variants by one assay. Immunoassay methods are well known in the art and generally comprise: (a) delivering a polypeptide comprising an epitope that can be linked by means of an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions that allow the formation of an antibody-antigen complex; and (c) determining whether the antibody-antigen complex comprising said polypeptide is formed. Polypeptides of the invention can be by synthetic means (for example as described by Geysen et al.) Or recombinantly, as described below. Particularly preferred palpeptides of the invention include those that span the lipidkine domain or that are within said domain, ie from amino acids 2326 to 2644 of sequence ID No. 2 or sequences substantially homologous thereto. Fragments in accordance with what is described above from this region are particularly preferred. The polypeptides and fragments thereof may contain amino acid alterations in accordance with the above defined, including substitutions at one or more of positions 2475, 2480 and 2494, which correspond to the positions of the rad3 substitutions described in the following examples. Preferred substitutions include D2475A, N2480K and D2494E. The polypeptides of the present invention can be used in similar culture systems in vivo or in vitro to study the function of ATR as a checkpoint gene. For example, truncated or modified ATRs (for example modified in the lipidkine domain) can be introduced into a cell to upset the normal checkpoint functions that occur in the cell. The polypeptides of the invention can be introduced into the cell by the expression in. site of the polypeptide from the recombinant expression vector (see below). The expression vector optionally carries an inducible promoter to control expression of the polypeptide. It is expected that the use of mammalian host cells will provide post-translational modifications (eg, mythiolation, glycosylation, truncation, stoning and phosphorylation of tyrosine, serine or threonine), as may be required to provide optimal biological activity in recombinant expression products of the invention. Such cell culture systems in which the polypeptides of the invention are expressed can be used in test systems to identify candidate substances that interfere with checkpoint functions or increase the checkpoint functions in the cell (cf. then). In an additional aspect, polypeptides of the present invention include the sequence ID protein No. 4 and fragments thereof from the region other than the fragment consisting of amino acids 713 to 1778. Particularly preferred fragments include those comprising residues 2082 a 2386 (the ipidkinase domain) and the leucine closure regions 298 to 347 and 576 to 591. Additionally, the fragment comprising the conserved region 1103 to 1111 is preferred. Such polypeptides and fragments can be made and used in accordance with described above. The invention also provides polypeptides substantially homologous with the sequence ID protein No. 4, and fragments thereof. In this context, a substantial homology is considered co or a sequence, which has at least one amino acid homology (identity) of 70%, for example 80% or 90%, in 30 amino acids with the sequence sequence ID No. 4 except in relation to the lipidkine domain and in the C-terminal portion (residues 2082 to 2386) where a? Substantial homology is considered to be a homology (identity) of at least 80%, preferably at least 90%, over 50 amino acids. C. Vectors The polynucleotides of the invention can be incorporated into a replicable recombinant vector. The vector can be used to replicate the nucleic acid in the compatible host cell. Accordingly, in a further embodiment, the invention provides a method for preparing polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and culturing the host cell under conditions that cause the replication of the vector. The vector can be recovered from the host cell. Suitable host cells are described below in relation to expression vectors. D. Expression Vectors Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that can provide expression of the coding sequence by the host cell, ie, the vector is a vector and expression. The term "operably linked" refers to a juxtaposition where the described components are in a relationship that allows them to function in a predicted manner. A control sequence "operably linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under a condition compatible with the control sequences. Such vectors can be transformed into a suitable host cell in accordance with that described above to provide for the expression of a polypeptide of the invention.

Accordingly, in a further aspect, the invention provides a process for preparing polypeptides according to the invention which comprises culturing a host cell transformed or transfected with an expression vector in accordance with that described above under conditions to provide the expression by the vector of a coding sequence encoding the palpeptides, and recovering the expressed polyps. The vectors can be, for example, plasmid vectors, viruses or phages provided with an origin of replication, ap proionally a promoter for the expression of said polynucleotides and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example, a gene for amphetamine resistance in the case of a bacterial plasmid or a gene for neomycin resistance in the case of a vector of a mammal Vectors can be used in vitro, for example, for the production of APN or used to transfect or transform a host cell. The vector can also be adapted to its use in vivo, for example in a gene therapy method. A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention. The cells will be chosen for their compatibility with said vector and can for example be bacterial, yeast, insect or mammalian. The polynucleotides according to the invention can also be inserted into the vectors described above in an antisense orientation in order to provide the production of anti-sense RNA. The antisense RNA and other antisense polynucleotides can also be produced by synthetic means. Such antisense polynucleotides can be employed in a method for controlling ATR levels or their variants or species homologs. Promoters and other expression regulation signals can be selected for compatibility with the host cell for which the expression vector is designed. For example, promoters of ligation include promoters GAL4 and ADH of S. cerevisiae, promoters nmtl and ADH of S. pombe. Mammalian promoters include the ionein metalot promoter which may be included in response to heavy metals such as cadmium. Viral promoters such as for example a large SV40 C-gen promoter or adenovirus promoters can also be used. All of these promoters are readily available in the art. E. Antibodies The invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof. The invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention. Monoclonal antibodies can be prepared by a conventional hybridoma technology using the polypeptides of the invention or polypeptide fragments thereof, as an immunogen. Polyclonal antibodies can also be prepared by conventional means comprising the inoculation of a host animal, for example a rat or a rabbit, with a polypeptide of the invention or peptide fragment thereof and recovery of immune serum. For pod <In preparing such antibodies, the invention also provides polypeptides of the invention or fragments thereof haptenized to other polypeptides for use as immunogens in animals or humans. Preferred antibodies of the invention may be linked to the human ATR protein with an affinity of at least 10 -sequences, preferably at least 100 times greater than the rad3 protein. Such antibodies can be obtained by routine experiments, for example by selecting regions of ATR protein with different sequences from the corresponding regions of rad3, making peptides comprising such sequences and using such peptides as immunogens. After the production of antibodies, the binding of said antibodies can be determined. Preferred antibodies of the invention include antibodies capable of selectively binding to the lipidkinase domain (according to the above) of the human ATR protein. In addition, antibodies that can bind the lipidkinase domains of human and lavage (S, pombe) with a similar affinity, but not the domains of the ATM family of proteins form a further aspect of the invention. Such antibodies can be prepared against peptides from the lipidkinase domains corresponding to regions found identical, or substantially identical, in the genes of yeast and humans. For the purposes of this invention, the term "antibody", unless otherwise specified, includes fragments of whole antibodies that retain their binding capacity for a target tumor antigen. Such fragments include the Fv, F (ab ') and F (ab') 2 fragments, as well as single chain antibodies. In addition, the antibodies and fragments thereof can be humanized antibodies, for example in accordance with that described in EP-A-239400. The antibodies can be used in methods for the detection of polypeptides of the invention present in biological samples by means of a method comprising; (a) providing an antibody of the invention; (b) incubating a biological sample with said antibody under conditions that allow the formation of an antibody-antigen complex; and (c) determining whether the antibody-antigen complex comprising said antibody is formed. Suitable samples include extracts from dividing cells, for example leukocytes or cancer cells including leukemia cells and solid tumors such as for example breast, ovarian, lung, colon, pancreas, testes, liver, brain, muscle and bone tumors. The antibodies of the present invention can be bound on a solid support and / or formed together in a suitable container together with suitable reagents, controls, instructions and the like. F. Assays The abrogation of cell cycle control points is a potential strategy for the development or design of drugs for anticancer therapy, both as a novel treatment and as part of a combination therapy to increase the specific toxicity of chemotherapeutic agents. current For example, alkylating agents such as nitrogen mustards »employ as chemotherapeutic agents that damage DNA in cells that divide rapidly, leading to cell death. The toxicity of such agents can be reduced by DNA repair and checkpoint mechanisms. The abrogation of these mechanisms will therefore increase the effectiveness of therapeutic compounds designed to damage DNA. The abrogation of the control point of. ATR is especially useful in cases in which cells have lost other damage-response genes or checkpoints, since these other genes may be able to complement the loss of ATR function in non-tumor cells, leading to a even greater increase in the effectiveness of the chemotherapeutic agent. The lipidkinase activity of ATR is an objective to develop anti- cancer compounds, since the results presented in the following examples indicate that the kinase domain is required for the ATP function. Accordingly, the present invention provides a test method for screening candidate substances for anti-cancer therapy, which includes: (a) providing a polypeptide of the invention that conserves the activity of lipidkinase and a substrate for said kinase, under conditions and with reagents such that the kinase activity acts on the substrate; (b) contacting said polypeptide and substrate with a candidate substance; (c) measuring the degree of decrease in kinase and polypeptide activity; and (d) selecting a candidate substance that provides a decrease in activity. The assay can be carried out in vitro, for example in the wells of a microtiter plate. Such a format can be easily adapted for automation, allowing the screening of a large number of candidate substances. The substrate can be a protein or lipid substrate of natural or synthetic origin on which the polypeptide of the invention will act. Typically, the polypeptides of the invention will phosphorylate the substrate. Any format suitable for testing can be used by those skilled in the art of testing. Typically, the polypeptide of the invention which retains a lipidq-inase activity will be bound to a solid support in the presence of a substrate and cellular and other components usually required for the activity. The labeled phosphate and a candidate substance will be added to the mixture simultaneously or sequentially in any of the orders. After a suitable reaction time. { usually some minutes but, in any way, a sufficient time to allow the phosphorylation of the substrate in the absence of the candidate substance), the amount of free phosphate is determined, for example by phosphate precipitation. Candidate substances that inhibit kinase activity will inhibit the uptake of free phosphate into the substrate and therefore where free phosphate is found is an indication of inhibition. Other test formats can be used by those skilled in the art. The candidate substances can be used in an initial screening, in batches of, for example, 10 compounds per reaction, and the compounds of these batches that exhibit inhibition will be tested individually. Suitable candidate substances and peptides, especially of approximately 5 to 20 amino acids in size, based on the sequence of the kinase domain, or variants of such peptides wherein one or more residues have been substituted in accordance with that described above. Peptides from peptide panels comprise sec-on or random sequences that have been varied consistently to provide a maximally diverse panel of peptides. Other candidate substances include kinase inhibitors which are small molecules such as for example compounds of cyclosporin and staurosporine, or other compounds commercially available in panels of small molecule inhibitors. Candidate substances that exhibit activity in in vitro sieves such as the foregoing can then be tested in in vivo systems, such as for example yeast or mammalian cells that will be exposed to the inhibitor and tested for checkpoint activity. We have also shown that Rad3 possesses a protein kinase activity. White Protein Kinase Rad-3 activity substrates can be identified by incorporating test compounds into assays to determine kinase activity. The Rad-3 protein is resuspended in a kinase regulator and incubated either in the presence or absence of the test compound (e.g., casein, histone H1, or appropriate substrate peptide). Phosphate molasses transferred by the kinase to the test compound are measured by autoradiography or scintillation. The transfer of phosphate to the test compound is indicative that the test compound is a substrate of the kinase. Agents that modulate the Pad-3 / ATR lipidkines or the roteinqui asa Rad3 activity can be identified by incubation of a test compound and can be immunopurified by Rad3 / ATR from cells that naturally express Rad3 / ATR, with Rad3 / ATR obtained from recombinant prokaryotic or eukaryotic cells expressing the enzyme, or with purified Rad / ATR, and then determining the effect of the test compound on the Rad3 / ATR activity. The lipidkinase activity of Rad3 / ATR or Rad3 protein kinase domains can be measured by determining the moles of 32P-phosphate transferred by the kinase from gamma 32-P-ATP either towards itself (autophosphorylation). ) or towards an exogenous substrate such as a protein or a lipid. The amount of phosphate incorporated in the substrate is measured by scintillation counting or by autoradiography. An increase in the moles of phosphate transferred to the substrate in the presence of the test compound compared to the moles of phosphate transferred to the substrate in the absence of the test compound indicates that the test compound is an activator of said kinase activity. Conversely, a decrease in the mole of phosphate transferred to the substrate in the presence of the test compound compared to the mole of phosphate transferred to the substrate in the presence of the test compound indicates that the modulator is an inhibitor of the substance. . i. pot. In a preferred assay, a Rad3-'ATR antibody bound to agarose beads is incubated with a cell lysate prepared from host cells to be expressed, Rad3 / ATR. The beads are washed to remove proteins that bind not specifically on the beads and the beads are then resuspended in a kinase regulator (with or for example 25 mM K-HEPES pH 7.7, 50 M. Potassium chloride, 10 mM of magnesium chloride, 0.1% Nopidet P-0, 20% glycerol, 1 M DTT). The reaction is initiated by the action of 100 μm of gamma-32P-ATP (4 Ci / mM) and an exogenous substrate such as for example lipid or peptide, and the reaction is carried out at a temperature of 30 ° C for 10 minutes . The activity of the kinase is measured by determining the mole of 32P-phosphate transferred to either the kinase itself or the added substrate. In a preferred embodiment, the na host cells have the endogenous P.ad-3 / ATR kinase activity. The selectivity of a compound that modulates the lipidkinase activity of Rad3 / ATR can be evaluated by comparing its activity in Rad3 / ATR with its activity, for example, on other related drugs with phosphatidin-1 inositoi-3 (PI-3). ) known. The combination of the recombinant Rad3 / ATR products of the invention with other kinase products related to PS-3 recombined in a series of i-dependent assays provides a system for developing modulators selective for the Rad-3 / ATR kinase activity. Similarly, the selectivity of a compound that modulates the protein kinase activity of Rad3 can be determined with reference to other protein kinases, for example protein dependent proteinase DNA or ATM. In addition, the demonstration that rad mutant, rad.D2249E (see examples) can act as a dominant negative mutant indicating its involvement in one or several protein complexes and such complexes themselves may be targeted for therapeutic intervention. We have shown, for example, that Rad3 can self-associate and associate with ATR.

Therefore, it is likely that Rad3 / ATR functions as chimeric molecules. Genes of human ATR or of R d of yeast mutants, or derivatives thereof which also do not have a Rad / ATP activity. they can be introduced into cells to act as dominant negative mutants.

Accordingly, if the expression of a dominant negative mutant (for example D2475A, N2480K or D2494E of ATR) in a tumor cell leads to an increased sensitivity to radiation this indicates that ATP. native continues to function and therefore remains the target of therapeutic agents. Proteins that interact with, including mutant protein complex components that change Rad3 or bi n ATR can be identified by the following assays. A first test contemplated by the invention is a two-hybrid screen. The two-hybrid system was developed in yeast (Chien et al. (1991)) and is based on a functional in vivo reconsti tion of a transcription factor that activates a reporter gene. Specifically, a polynucleotide encoding a protein that interacts with Rad3 / ATR is isolated by: transformation or appropriate transfection of host cells by a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having DNA, a binding domain and an activation domain; the expression in the host cells of a first hybrid DNA sequence encoding a first fusion of part or all of Rad3 / ATR and either the DNA binding domain or the activation domain of the ancription factor; the expression is the host cell of a library of second hybrid DNA sequences encoding a second fusion of part or all of putative Rad3 / ATR binding proteins and the DNA binding domain or the activation domain of the transcription that is not incorporated in the first merger; detecting binding of a Pad-3 / ATR interaction protein for Rad-3 / ATR in a particular host cell by detecting the production of reporter gene products in the host cell; and the isolation of second hybrid DNA sequences encoding the interaction protein from the particular host cell. A lexA promoter for inculcating expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the DNA binding domain of le? A and the transactivation domain SAL.4, is currently preferred for use in the assay. and yeast host cells. Other assays to identify proteins that interact with Rad3 or ATR may involve the immobilization of Rad3 / ATR or a test protein, detectably labeling the non-immobilized binding partner, incubating the link partners together and determining the amount of united marker. The bound label indicates that the test protein interacts with Pad3 / ATR. Another type of assay to identify proteins that interact with Rad3 or ATP. includes the immobilization of Rad3 / ATR or a fragment thereof on a solid support coated (or impregnated with) a fluorescent agent, labeling a test protein with a compound capable of exciting the fluorescent agent, contacting immobilized Rad3 / ATR with a labeled test protein, detecting light emission by the luorescent agent, and identifying proteins interacting as test proteins that result in the emission of light by the fluorescent agent. Alternately, the protein interacts. You can be immobilized and you can mark Pad3.'ATP in the trial. Compounds that modulate the interaction between Rad3 / ATR and other cellular components can be used in methods for the treatment of cancer »For example, if a particular form of cancer results from mutation in a gene other than ATR such as the p53 gene, an agent that inhibits the transcription of ATR enzymatic activity and therefore the control point of the G2 cell cycle can be used to make cancer cells more susceptible to chemotherapy or radiation therapy. The therapeutic value of such an agent lies in the fact that current radiation therapy or current chemotherapy in most cases does not exceed the ability of the p53 mutant cancer cell to detect and correct the DNA damage imposed as a result of the treatment. As a result, a cancer cell can simply repair DNA damage. Modulation agents of the invention can therefore be adjuvants of chemotherapy and radiation or they can be directly active as chemotherapy drugs themselves. Assays for identifying compounds that modulate the interaction of Rad3 / ATR with other proteins may include: the transformation or transfection of appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor that has a DNA binding domain and an activation domain; the expression in the host cells of a first hybrid DNA sequence encoding a first fusion of part or all of Rad3 / ATR and the DNA binding domain or the activation domain of the transcription factor; the expression in host cells of a second hybrid DNA frequency encoding part or all of a protein that interacts with Rad3 / ATR and the DNA binding domain or the activation domain of the transduction factor not found incorporated in the first merger; evaluating the effect of a test compound on the interaction between Rad3 / ATR and the interacting protein by detecting the binding of the interacting pratein with Rad3 / ATR in a particular host cell by reducing the production of product cié reporter gene in the host cell in the presence or absence of the test compound; and identifying co-modulation compounds or test compounds that alter the production of the reported gene product in comparison to the production of the reporter gene product in the absence of the modulation compound. Currently it is preferred for your us. in the assay a lexA promoter for activating the expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the DNA domain of lex and the GAL4 t-ion ion domain, and yeast host cells. Another type of assay for identifying compounds that modulate the interaction between Rad3 / ATR and an interaction protein includes the immobilization of Rad3 / ATR or an interaction protein of Rad3 / ATR, detectably labeling the non-immobilized binding partner. , incubating the binding partners together and determining the effect of the test compound on the amount of bound marker where a reduction of the bound label in the presence of the test compound compared to the amount of bound label in the absence of the test compound indicates that the The test agent is an inhibitor of the Rad3 / ATR interaction with the protein. Conversely, an increase in the binding in the presence of the test compound compared to the amount of bound label and absence of the test compound indicates that the putative modulator is an activator of the Rad3 / ATR interaction with the protein. However, another method concentrated by the invention to identify compounds that modulate the bond between Rad3 / ATR and a interacting protein includes the immobilization of Pad-3 / ATR or a fragment thereof on solid support coated Co well impregnated) with a luorescent agent marking the interacting protein with a compound capable of exciting the fluorescent agent, counting the immobilized Rad-3 / ATR with the interacting protein labeled in the presence and absence of a test compound, detecting the emission of light by the fluorescent agent, and identifying modulating compounds or test compounds that affect the emission of light by the fluorescent agent compared to the emission of light by the fluorescent agent in the absence of the test compound. Alternatively, the interacting protein with Rad3 / ATR can be immobilized and Rad3 / ATR can be labeled in the assay. We have shown that Rad3 interacts with ATR. Therefore, the aforementioned assays can also be used to identify compounds that modulate the interaction between Rad3 and ATR where the inter-acting protein described in the test methods is either R.ad-3 or ATR. We have also shown that Rad3 can bind on itself, which strongly suggests that ATR. You can also unite about yourself. Therefore, the aforementioned assays can also be used to identify compounds that modulate Rad3-P.ad3 interactions and ATP-TR interactions. Such compounds could be used therapeutically to upset the ATR-ATR interactions and increase the sensitivity of tumor cells to chemotherapy and / or radiotherapy. Accordingly, the invention provides a test method for screening candidate substances for a cancer therapy comprising: (a) (i) incubating a polypeptide of the invention with another polypeptide of the invention, which may be the same or may be be different from the first polypeptide, under conditions that allow the first polypeptide to be linked to the second polypeptide to form a complex; (i i) contacting the complex formed in this way with a candidate substance; or (a) incubating a polypeptide of the present invention with another ip polypeptide of the invention, which may be the same or different from the first polypeptide, under conditions that allow the first polypeptide to be linked over the second polypeptide to form a complex and in the presence of a CA d idata substance; 15 and (b) determining whether the candidate substance inhibits the binding of the first polypeptide on the second palpeptide and (c) selecting the candidate substance that inhibits the binding of the first polypeptide on the second polypeptide. ? < Preferably, the first polypeptide and the second polypeptide may be different from each other. The first polypeptide and the second polypeptide can both be ATR, or they can both be Rad-3, or one can be ATR. and one can be Rad3, or derivatives of either ATR or Ra 3 that retain their binding activity. When both polypeptides are ATP or Rad3, preferably two distinguishable forms of ATR / Rad3 would be employed in these assays. They can be distinguished, for example, by labeling any of the polypeptides. Examples of labels include radioactive labels, epitope labels or other palpente labels such as glutathione-S-transferase. For example, one form of Pad3 may have one form of epitope marker, and the other form could be a different epitope marker, allowing it to be distinguished immunologically in such a way that the binding of one _- > The other may be determined quantitatively or quantitatively. In a preferred method, the first pol. Eptid can be immobilized, for example, on pearls of agarose or a solid support, and the second polypeptide can be in a free solution. The link is then determined using methods described above and well known to those skilled in the art. Likewise, the present invention encompasses antibody products by emp3o, monoclonal antibodies and pe1 1 ona le =, single chain antibodies, chimeric antibodies, antibodies infected with CDP and the like) and OGGHS binding proteins (such as those identified in the previous tests) that are specific to the protein domain or Pad3 mass or the lipidkinase domains of Pad3 / ATP. Protein binding can be de-activated, using isolated natural or recombinant enzymes. The binding proteins are useful, in turn, for purifying recombinant and natural enzymes and for identifying cells that produce such enzymes. Assays for the detection and quantification of proteins in cells and in fluids may include a single antibody substance or multiple antibody substances in a "sandwich" assay format. The binding proteins are also obviously useful for modulating ie blocking, inhibiting or stimulating) enzyme / substrate interactions or enzyme / regulator. Modulators of Pad3 / ATF can affect its qumasa activity, its when it is ionized in the cell, and / or its interaction with members of the pathway of cell cycle control points.

Selective modulators may include such as, for example, polypeptides or peptides that specifically bind to Rad3 / ATR or Nuclean acid from Pad3 / ATR, and / or other non-peptide compounds eg, organic or isolated organic molecules) which? They specifically react with Ra / ATR or b i in S I o nucleic of Pad3 / ATP. Forms mutaptes e Rad3, 'ATP que. fec,. The ennmitic activity of either wild type or Rad3 / ATR is also contemplated in the invention. Thus, libraries combine ori s, peptides and peptide mimetics, defined chemical entities, oligonucleotides, and libraries of natural products can be screened for their activity as modulators of the Rad3 / ATR kinase activity and Rad3 / ATR interactions in such assays. co or those described above. F. Therapeutic uses Modulators of Rad3 / ATR activity, including inhibitors of lipidkinase and protein kinase activities, can be used in anti-cancer therapy. Particularly, it can be used to increase the susceptibility of cancer cells to chemotherapy and / or radiotherapy by virtue of their ability to disrupt the regu functions. atona.-, of the cell cycle of Pad3 / ATP. Accordingly, the invention provides for the use of compounds that modulate the Rad3 / ATR activity, identified by the screening assays described above, in a cancer treatment method. In one embodiment, said compounds can inhibit the lipidkinase activity of Pad3 / ATP and -'or the protein kinase activity of Pad3. In another embodiment, said compounds can inhibit intimations. between ATP and itself, 'to ATR and other proteins ifilerjc tuantt?' r, which may, for example, normally be part of a complex of proteins m ?timépco. It is understood that the term "compound" in this context is also referred to. the candidate substances selected in the assays described above. Typically the compounds are formulated for chemical administration by mixing them with a pharmaceutically acceptable carrier or diluent. For example, they can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration. Preferably the compound is used in injectable form.

Direct injection into the patient's tumor is beneficial because it makes it possible to concentrate the therapeutic effect at the level of the affected tissues. It can therefore be mixed with any pharmaceutically acceptable carrier for an injectable formulation, preferably for a direct injection at the site to be treated. The pharmaceutically acceptable vehicle or diluent can be, for example, sterile or isotonic solutions. The dose of compound used can be adjusted according to several parameters, especially in accordance with the compound used, the age, date and condition of the patient to be treated, the mode of administration used, the pathology of the tumor and the clinical regimen used. Co or guide, 3rd quantity of compound administered by injection e = > suitably from 0.01 mg / l to 30 mg / kg, preferably from 0.1 m / l to IO and / tg. . = .- 5 ways ej adminis ra »: i n and descriptions described have only purpose to be a guide since an experienced doctor can easily determine the optimal route of administration and the optimal dosage for a given patient and a specific condition. The compounds to be administered may include polypeptides or nucleic acids. The nucleic acids can encode p.1-peptides and can also encode antisense constructs that inhibit the expression of a cellular gene. The nucleic acids can be administered, for example, by lipofection or by viral vectors. For example, the nucleic acid can be part of a viral vector such as, for example, adenovirus. When viral v uers are employed, in general, the administered dose is between 10,000 and 100,000,000,000,000 pfu / ml, preferably between 1,000,000 and 10,000,000,000 pfu / ml. The term "pfu" "plaque forming unit") corresponds to the infectivity of a viral solution and is determined by infection of an appropriate cell culture and measurement, usually after 48 hours, of the number of plaques of infected cells. The techniques for determining the culture of pfu of a viral solution are well documented in the literature.u ra. All types of cancers can be treated by these methods, eg, leu > - «, r, á 3, and solid tumors such as for example or or of ma, ovaries, lung, colon, pancreas, tesicles, liver, brain, muscle and bones. Preferably, the tumor has a normal ATR function. Description of the Drawings Figure 1 The relationship between ATR, rad3, mei-41, MECI, TEL1 and ATM A. Global structures of ATR, Rad3, Mei-41, Meclp, Tellp and ATM. Caption: empty square - Domain Rad3; shaded boxes - kinase domain B. Dendrogram based on sequence alignments generated by the Clustal method (PAM250) using the DNAstar program. rad4 / ESRl / mei-41 / ATR relate more closely to each other than to ATM and TEL1. Rad-3 and ATM sequences are available in the EMBL database. The following examples illustrate the invention. Example 1 The rad3 gene of S. pombe is one of six genes absolutely required for the control times of the structure of DNA in S. pambe (Al- hodairy and Carr, 1992; Al-Khodiry et al. 1994). A sequence representing part of the rad-3 gene was published by Seaton et al. (1992). When trying to clarify the structure i. nt ron / e--: t rum of this gene, we identify -anormalities of sec? enc i-amiento both at the 5 'end and at the. 3 'end. We sequence the entire gene (see Experiments) and we found that rad3 can encode a product of 2386 amino acids. The C-terminal region contains the consensus sequences typical of a subclass of kinases known as 1 ipidquinases, the founding member of said subclass being the pllO catalytic unit of PI3 kinase (Hiles et al., 1992). A clone of truncated rad3 that does not have the amino terminus and the kinase region has been trimmed to complement the mutant for rad3i: pR3Hl .0 rad3 genetic disorder (Jiménez et al., 1992). This disorder mutant does not remove the potential kinase domain. To clarify the function of this domain, we created a null mutant by gene replacement. This mutant has the amino acids 1477-2271 of rad3, i c: showing the consensus domain of kinase, replaced by ura4 +. This strain, rad3.d, has identical checkpoint defects and radiation / hydroxy sensitivities identical to the rad3.136 mutant (Nasim and Smith et al., 1975), and the rad3i disorder mutant: pR3Hl .0 original (Jiménez et al., 1992: Seatan et al., 1992) (data not illustrated). We created three separate point mutants in the putative radon qui nase domain and used them in gene replacement experiments to construct mutant strains > ::: null s of what are defined. The three strains, rad-3. D2230A, rad3.N2235. and rad-3. D2249E have identical phenotypes to the rad-3.d null mutant (data not shown), suggesting that the kinase activity refers to the rad-3 function. In view of our findings, an interpretation of the results of Seaton et al. (1992) and Jiménez et al. (1992) is that the partial clone can illustrate a strategic comparison between the truncated gene carried in plasmid and a partial genomic removal that retains the kinase function. Such an interpretation would be consistent with Rad3 acting as a day or multimer. When the null allele of those cells rad3.D2249E was moderately over-expressed in wild type cells or the control of a modified nmtl promoter (Maundrell, 1990), it reversed an extreme sensitivity to radiation, tested by means of band test. of UV rays, and acted as a dominant negative mutant (data not shown). When the same null construct of kinase was expressed at a higher level, it inhibited growth (data not illustrated). Examination of the cells indicates that the division proceeded very slowly, and in a smaller cell size, wild-type cells and cells containing an empty vector were approximately 15 microns apart, while cells overexpressing rad3 and rad3. D2249E are divided into approximately 11.2 microns (data not illustrated). In 3. pombe, this indicates you had an immense a n > .: e de i o is "The homologue of human rad-3, ATR To identify a human form of rad-3, a combination of methods was applied. Through these approaches, we cloned the entire coding region of a human gene (see materials and methods), which we have named ATR (related to ataxia and rad). ATR can encode a protein of 2644 amino acids much more related to the genes of rad3 genes of S. pombe, ESP.1 of S. cerevisiae (ato and Ogawa, 1994) and mei-41 of D. melanogaster (Hari et al., 1995 ) than with the human ATM and Tell proteins from S. cerevisiae (Savitsky et al., 1995; Greenwell et al., 1995) and is likely to be the true homologue of rad-3.ESRl is allelic to the point-of-control mutants me l / sad3 (Alien et al., 1994; Weinert et al., 1994) that have a phenotype equivalent to rad3. ATR is more closely related to the human ATM control point gene, which contains the domain of lipidkinase put ivo of C-terminal and which has a similar global structure. The sequence alignments clearly demonstrate that the rad3 / ESR1 (MECI / SAD3) / mei-41 / ATR genes are more closely related to each other than to ATM or TEL1, and that ATM is more homologous to TEL1 (Figure 1). The ATM gene is expressed in several tissues (Savitsky et al., 1995). In S. cerevisiae, ESR1 shows a low level of expression in mitotic cells but is rapidly induced during meiosis I (Kato and Ogawa, 1994). Using a Northern blot analysis, we show that ATR is also weakly expressed in many tissues but that it is found most highly expressed in testes (data not shown). Since the proteins of ATR, rad3 and Esrlp are more highly related to each other than to ATM, the highest expression of ATP. in the testes it is consistent with the observation that Esrlp plays a role in meiotic recombination (Kato and Ogawa, 1994). Using FISH analysis and PCR, we mapped ATR to chromosome 3q22-3q25 (data not shown). This region is not related to syndromes known for its. carcinogenic probability. In order to investigate further the possibility that • rad-3 act as uí. timer, creates two separate marked rad-3 total length constructs in inducible vectors based on pP.EP. In one of them, Rad3 is transferred with two mye epitope markers at the N end while in the other these are substituted by a triple epitope marker HA. When both constructs are expressed together wild type cells, it is possible to coprecipitate the HA-labeled Rad-3 with the specific antibody for myc and the Rad3 labeled with myc with the specific antibody for HA (data not illustrated). This demonstrates that, in iv., The P.ad-3 protein can self-associate and is fully consistent with the complementation data of Jiménez et al. (! 992). Even when the ATP gene. could not complement the rad3 mutant phenotype, we investigated the ability of ATR to form a protein complex with S. pombe rad3 by expressing both ATR and Rad3 from S. pombe labeled with myc in the same yeast cells. Using an anti-ATR antibody (which does not precipitate S. rad3 or be, see materials and methods), we were able to coprecipitate the yeast protein. We were also able to precipitate the human ART protein with antibodies specific for myc that recognize the Rad3 from S. pombe (data not shown). These data suggest that human and yeast protein can form a heteromeric complex, which supports the point of view, based on the similarity of sequences, of a close functional relationship between these homologs. Rad3 proteins have an associated kinase activity Since the mutagenesis experiments suggest that the kinase activity of? the proteins of P.ad-3 in vivo seems to be essential for its. function, investigate this activity more thoroughly. Using radionic cells4 of S. pombe expressing Rad3 from S. pombe labeled with HA, we were able to detect a significant activated protein kinase that precipitated with specific antibodies for HA only? when P.as3 is induced and does not change after irradiating ion (data not illus- trated). This activity, specific for P.ad3 or coprecipient kinase, seems to reflect the radionuclide phosphorylation, since the main band above 20O kD which is phosphorylated can be detected by Western analysis with anti-DNA antibody. -HA (data not illustrated). Attempts to identify suitable in vitro substrates such as, for example, ina-basic honey protein, RP-A and several purified S. pombe control point proteins have not been successful to date. When an IP in vitro kinase assay is performed with cells overexpressing a D2249E "null kinase" version of Rad3, the associated kinase activity precipitated by antibody specific for HA is significantly reduced (data not shown). There are many possible explanations for this situation. The activated kinase measure could directly reflect the Rad3 activity. In this case, the residual activity seen by P.ad-3 of killed kinase could reflect the fact that it is not unknown for the mutation equivalent of D to E in another protein kinase to produce a biologically inert protein with a residual biochemical activity in vitro. Alternatively, the kinase activity with phosphorylated rad3 may be due to associated proteins, and these may interfere less effectively with the mutant protein D2249E. Commensurate The checkpoint pathways that control the progression of the cell cycle after damage or interaction with the DNA in the individual events that comprise the cycle are of considerable importance in maintaining genetic stability and can be considered as pathways that suppress the tumor genesis. Several tumor suppressor genes are intimately involved in their sets of checkpoint pathways (reviewed in Hartwell and Kastan, 1994), particularly those that affect the transition from 61 in S phase and assignment to the cell cycle. The convergence of the two yeast model systems for control points clearly indicates that the genes involved in these pathways are conserved. Our work extends this conservation to etazoa cells, and clarifies the relationship between rad3, ESR1 (MECI / SAD3), mei-41 and the ATM gene. In this paper we show that the correct sequence of the rad-3 gene places its. product in the family of protein / 1 ipidkinasas related to ATM. Overexpression of defective rad3 for utapte kinase in S. pombe causes a dominant negative phenotype, suggesting that Rad3 acts as a member of a protein complex whose integrity is necessary for the function of the control point. This is consistent with the observation that the radl, rad9, radl7, rad26 and husl deletion mutants have all phenotypes that can not be distinguished from rad3.d iSheldrict and Carr, 1993). Unexpectedly, unlike the remaining control point rad genes, a high level of envelope: - pressure of wild type or mutant rad3 alleles inhibits cell growth and causes the occurrence of mitosis in a reduced cell size, which indicates a premature entry into mitosis. This phenotype "is small" is not observed in the null mutant, and this may indicate an interference in a second path whose function splices the function of Rad3 and acts to inhibit mitosis. A candidate for said route is ATM / TEL1, which has been shown to have some splice functions with the ESR1 (MEC1 / SAD1) route (Morrow et al., 1995). The ATM structure is related more closely to the Tellp, which is involved in the maintenance of telomere length (Greenweel et al., nineteen ninety five). However, the ATM function also seems to be related to the function of the products of R ad-3 / Esrlp / mei-41. After the initial discovery according to which the ATM gene and its sequence relationship with the rad3 / ESR1 and TEL1 gene, it is not clear if, as in many cases in yeast, the gene has been duplicated and changed in yeast or if the Two yeast proteins defended their conserved families of closely related genes. The significant finding of this work is the identification of a human gene, ATP, plus it is re-related to rad-3 / ESR.l / mei-41. This defines two related subfamilies of control point is ruc ture 1 different entities of protein / 1 ip kinases that are conserved in eukaryotic evolution. Even though the proteins in these two subfamilies may have some splicing functions, they probably control different processes. For example: the rad3 subfamily in yeast controls all the DNA damage control points Gl and G2 in response to both UV and ionizing radiation, and the S phase control point that avoids mito'.i- after inhibition. of rp ica ión ÍA1- hodairy and Caír, 1992, Alien et al., 1994, Ule inert et al., 1994). In contrast, A-T cells have apo- lymic responses to a narrow range of DNA-damaging agents including ionizing radiation, hleo- icine, and neocrostatone, which produce strand breaks in DNA as a consequence of a radical attack. The responses to UV rays and to most chemical carcinogens is normal, as well as 3. is the response 3 the inhibition of DNA synthesis. It is possible that some or all of the remaining DNA damage control points and the S phase control point are controlled by ATP. Procedures of the Cepa experiment, and the standard genetic techniques, growth conditions and means for S. po be described in Gutz et al. 1974). the spOll strain of S. pn beura4 »D18, leul.32 ade6.704 h) has been recently described (M? rray et al., 1992). The piSswido pSUB41 was presented by S. Subra to íSeaton et al. 1992). Cloning of ra >;: 13 of S. pombe A Kpnl fragment of 4.0 tb was removed from pSUE 1 and sequenced in both directions to obtain the 5 'rad3 sequence. The 3 'clone was identified from a genomic library (Barbel et al., 1992) by colony hybridization using a 3' cIE 1 1 -b probe derived from the published rad3 sequence, and in both directions. In this way, the sequence of the entire rad3 gene was assembled. Muclantes rad3 nulos and "de kinase dead" A rad3 construct, where the 794 amino acids between aal477 and aa227_i including the domain of qmnas.) Were replaced by an ura4 + gene, was created using 13 mythology described in Barbel et al. 1992). A linear fragment of this was used to transform spOlt to uracil prototroph and the integration of a single copy at the rad3 locus was reviewed by Southern plot. To create the specific site of mutations from qum to null, a Ba HI-SalI fragment of 3.01 tb C-terminus of rad3 was mutated with either: GTTTTCGCCGGGCGCGCTCCCAAACCCAA, B: TTCATCAAACAATATCTTTTCGCCAT66CG, or C: CAAAAAGACAGTTGAATTC6ACATGGATAG) for the purpose of trodu go either the mutation of D2230A, N2235 or D2249E between the quasan domain. Analogous changes had previously been employed in the analysis of VPS34 of PI3 quina .. of S. cerev isiae (Sr.hu et al., 1993). These fragments were then used to transform the null mutant rad3.d and replace it with the > They were selected because of their ability to grow in media containing FOA íGri m ei al. 19SS). All strains were checked by means of Southern blot. The full length expression constructs of rad3. D2230A were created in pREFl and pPFp41 (Maundrell, 1990) by standard subcloning emp 3 with the introduction of an Ndel site in the ATG and removing three internal Ndel sites. Radiation sensitivity bandwidth test UVW The expression of REP] (high) and PEP41 < intermediate) was induced by the absence of tiam to during 18 hours before placing in dish, it. plates were irradiated ..on a gradient of UV do.s on the plate from 0 to 300 Jm-2 according to the settings in a Str Lagene St to 1 irt! - er. Cloning and Expression of ATP To isolate an appropriate probe to identify cDNAs that correspond to a homologue of human rad3, degenerate ologonucleic leotons were designed against the amy no r- 1 two LGIGDPH (5 'oligo; oDHIG) and HVDFíD / NíC í3 'aligo; oDH-1 /,) of Rad3 / Esrlp. We were incorporated into positions of degeneration, and primers were linked with Ba HI (DH) and EcoPI (oDHl) to facilitate cloning. The DNA sequence analysis of the polymerase chain rearming product of approximately 100 bp obtained from the amplification of cDNA of peripheral blood leukocytes demonstrated a significant similarity with MFCl / rad3. This sequence was pooled to synthesize a non-degenerate primer (oDH-23; GACGCAGAATTCACCAGTCAAAGA rCAAAGAG) for polymerase chain reaction with an additional degenerate primer (HD17) designed against the amino acid sequence KFPP (J / V) (L / F) Y (Q / E) WF of Pad3 / E ^, rlp. The 174 bp product of this reaction was used discretely to screen a macrophage cDNA library. Four positive clones were isolated at the helper of approximately 3 b). In parallel, searches in database with rd3 of S. pombe of total length denial of the data of EMBL praporc lonó a clone of human cDNA, HSAAADPDG, co or homologue picdencial of rad3, if a change of single frame was allowed in the sequence of 233 bp. This sequence of 233 bp is contained within a range of 1.6 kb obtained from Dr. N. Affara, Human Molecular Genetics Research Group, University of Cambridge, Peino Unido. The entire clone (1.6 tb) was sequenced and found within the cDNA clones identified by degenerate poly-eraser chain reaction and library sieves. To identify the entire gene, experiments were carried out on the reaction of the PACE polymerase chain in cDNA derived from APN from placenta and ti mus using the proportional instructions.; jn a set of Clontech Marathon elements. Specific primers for genes were derived from the cDNA clones. From these experiments, a cDNA sequence of 8239 bpi was assembled with an internal OPF of 2644 amino acids, a 5 'non-coding region of 79 bp, a 3' non-coding region of 194 bp and a poly A-tail. - Parle: - of the sequence were determined only by polymerase chain reaction To avoid errors, clones with a minimum of 3 independent polymerase chain reactions were sequenced in both directions The sequence of 233 bp >corresponds to the sequence of nucleotides 6809 to 7042 234 nt in total) of sequence ID No.l except for a single base removal in position 6942. This sequence encodes amino acids 2244 to 2320 Sequence ID No. 2. The insert sequence of "1.6 kb" corresponds to nucleotides 5725 to 71 4 1353 nt) of sequence ID No. 1, and encodes amino acids 1892 to 2340 of sequence ID No. Hybridization Northern blot: a polymerase chain reaction product of 1.3 l < b was amplified in the presence of 32P-dCTP using primers of 279-3 íTGGATGATGACAGCTGTGTC) and 279-6 ÍTGTAGTCGCTGCTCAATGTC). A nylon membrane containing 2 pigs of po (A) RNA + fractionated pior size from a variety of human tissue source (Clontech Laboratories) was probed according to the manufacturer's request except that the final wash was performed at a ßC temperature instead of 5"C to minimize the possibility of cross-hybridization with related sequences ATP Mapping We mapped the ATR gene on chromosome 3 by a combination of assays based on in situ fluorescent hybridization and polymerase chain reaction (PCP) A FISH analysis using a cDNA clone identified the ATP gene on chromosome 3, on posit ion q22-23, and the polymerase chain reaction analysis also identified ATP on chromosome 3. Two initiators ioATR23 .6ACGCAATTCACCAGTCAAAGAATCAAAGAG and oATP26: TGGTTTCTGAGAACATTCCCTGA) that amplify a 257 bp fragment of the ATP gene were used in DNA derived from hybrids of ce Somatic human / rodent cells containing several panels of human chromosomes available from NIGMS Human Genetic Mutant Cell Pepository (Drwinga et al. 1993). A polymerase chain reaction was used with the same primers to subclass ATR in a specific region of chromosome 3. The tempers for these amplifications consisted of DNA samples from patients with truncations along chromosome 3, Leach et al. . 1994).

IP uncoupling assays) qumasa with Pad3 The human ATP and rad3 genes of S. pombe were cloned into e.-pressure vector of pREP41 for complementation studies. To label the proteins, versions of these vectors containing sequences of end-marker N in frame, whether a double myc marker or a triple HA marker, were used. 95). The labeled proteins were expressed by culture in non-amino media (Martinard, 1990). The yeast cells were used in lysis buffer (25 mM Tris.Cl, pH 7.5, 60 mM B-glychofosph, 0.1 M Na3VG4, IV, Triton X-100, 50 M MaCl, 2 mM of EDTA, 50 M NaF, 1 mM phene fluoride Ime 11 ul f or lo (PSMF), 5 μg / ml leupeptin, 5 μg / ml aprotimna, 1 mM DTT) by the addition of glass peeries followed by rubbing on a shredder for 2 minutes. For IP's, 300 pigments of total protein were incubated on ice with the api-opted antibody for 30 minutes and the immune complexes were precipitated by mixing with G-protein beads for an additional 30 minutes at a temperature of 4 ° C. For trials of which, the immune complexes were washed 4 times with a Li-regulator, once with a kinase regulator (35 M of Hpes, piH 7.7, 50 M of KCl, 10 M of MgC12, 0.17, of NP-4). 77A of gl irerol; t M of DTT), and incubated in kinase regulator with 10 μM of ATP (50 Ci / mmol)) for 25 minutes at a temperature of 30 ° C. The reactions were stopped with 20 ul 2X Sample regulator SDS before separation in polyacryl 1 to 6 * 4 gels.RTI3 IPs contained several phosphorylated products, including one that co-interacted with the Pad3 pyrotein itself in Western analysis. Al-Khodan-y, F., and Carr, AM U992). DNA r-epa go mutants defining G2 checppai nt pathways in Sch i zusaccharomyces pombe. (DNA repair killers that define a G2 control point path in Sch i zosaccharo yces pombe) EMBO J. 11, 1343-1350, Al- hodairy, F., Ftttou, E., Sheldrict, S.S., Gpffilhs, F, JF, Leh ann, AR and Carr, AM, 1994). Identification and charac ter of new elements involved in chetpioints and feedbaek controls in fission yeast. (Identification and characterization of new elements involved in control points and controls of retreat 3 ímenta i ón in fission yeasts) Mol. Biol. Cell 5, 147-160. Alien, J.B., Zhou, Z., Siede, W., Fpedberg, E.C. and Elledge, S.J. (1994) The SAD1 / PAD53 protein kinase controls multiple checl-points and DNA damage--? nduced t ranscr íp 11 on in yeast. (Prole i qui nasa SAD1 / PAD53 controls multiple control and transcription sites induced by DNA damage in yeast) Genes Dev. 8, 2416-2428.

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Sequence Information Sequence ID No. 1: ATR sequence 451 T (* ACTttTCT6pCApApAm < TITpAA A ^ 525 526 ApA AACTTTp (and \ ^ pG (_ ACCTCWT lAAG ^ 600 601 CATGAGC ^ TT AAGTWpAWTtWC TGGGATA 675 676 GCAAAAmAGAAmApGAAGTCACXpApM 750 751 GCAAGAACTOTACmGGCAGATAGipTGT ^^ 825 976 AAAGACACTAmcam &WGta ^^ 1050 1051? CTsGTGTCATG < »AGAC < £ TGTGsC ^^ 1125 1201 TAmGTAAAGCTCTmGG? TGTCCTT < ^ TT ^ 1275 1351 TC? TO ^ TATCACCCAAAAGGCGTCGra ^ 1425 1426 O ^ pAMCATGTGGACATGAACCAAAAliAl ^ 1500 1501 GAmCCCp (i4ATACAGTGGapA ACWTOT ^ 1575 1576 GACTGCTsGTGTACTGpttpGpCTCATCAAAACA.Cy ^^ 1650 1651 CAAGAAGAAACXpCTGTAGTGATAACpGGATGT ^^ 1725 1726 mGpAGAATCTGpOffiAAACTí ^ CTGGAG ^ 1800 1801 pATAT (iCAACTAAAC? GpCAm (iAAGATC? ^^ 1875 1876 pApCCCApCTG? T (_ATGGCTGmAAAGpGACCACAmGCXGCT TCpCTAACApAAGCTGTAG ^ 1950 2026 TGAGTOttGMCAGCAGmACAACTG ^^ 2100 2101 ATTTTTTATCpApGCAG AGCAGMpcpGTAA AGAGpCCCAA? PcpAT ^^ 2175 2176 pCTtVVWpGTCAAÍ? AAAG mGCpaATACpGGTCAACTTGTCTGTACTCpW ^ 2250 2326 TCMCATGAATGpCATCpCTCAACTAAAAGOT ^ 2400TCCAGTAAMCp &mttTAGATMT ^ 2475 2476 AACAGATGTAAAAGCAGpcpGGAAC ApAMmM 2550 2551 TAGTGClAAATATCAAGCACATApGGAATCm 2625 2626 MTGAAGGAAGCATATACACATGCCCAAATATC ^^ 2700 2701 GGATApGGAAGGGCCGCAAAAG (y ^ TTT (^ ACC? TpGC? CTCpACACp ^ 2775 2776 GTCAGCATCTGTCTCTGGAGCA (^ TACA (SW ^ 2850 ^ 2926 3000 TC iMTACTCCATGCCAGMTGCTGACGTGCGAAMÜWGATGIGGCTCACCAGWlAGA TGFI 3001 GpGTCTGAAApGCCAACGTTp (XAC CCTGATCp TCGTTpcpACTAGGACApACAAGpCTACl 3075 3301 ApGCATMTGAApATTGCTGOTApGGA { W CTATCAACAGGTTTpMT8 CT 3375 3601 (^ TGACCACACTGAGA? CTCGCmCGApCAA (_GATGAT ^ 3675 3751 CATCCAGCaAAA6AAACTGCA (ATCTTCCACTACCTCATMTT (lAAAA ^^ 3825 3901 GAaTCTGAGAGCACTGATOTCAGACAACTCpCAGCT CTATGAAGGCCAT ^ 3975 3976 TCGTApCAT (iCTCpACAA (XTTTiAAGGAAACCTTGTATAAAA ^ 4050 4051 CAGTGAAACACTAGAACCTApAmCACAGp (^ 4125 4201 TG * AAs (* AG »WWatmAW 4275 4351 GCTp ApATGAC GTAGAGACMTGGAGACCAACGGCCCAGG 4425 4426 TOTCGGGAAATACTAGA? CCTttTCTAMTAC ^ 4500 4576 TAO * GGpCGACATWTCpa AGTAA TTpCAaT (rrGTAGCApAT ^ 4650 4876 GAAAGCTGAllAAATGTCCACACAGCA TCAAACAGA ^ 4950 4951 AGACTATCAGACTGIAACíXGTmCIAGACCTC? Itf 5025 5026 AGWACACAttAGCTGTAATGCACmCiAATCAmAp ^ 5100 5101 ATimACAGAMTTGTATtt7GaATGCATGAACsG ^^ 5175 5176 ACCATCTsAAAAGAACAGATCCp < lAACATGAAAa ^ 5250 5251 GGCTApCAGCTAGAACCAGACOGAT pCApATCATGGTGTAGTAAAGTCCATGpAÍ ^ 5325 5326 G7CTACTGpATCAC7CAGG7GAA7GGAG7GCATGCTAACA ^ 5400 5401 AGTGGAA «GmGGAAApG7CACAGTGGGAm (3GTTiGAAA ^ 5475 5551 ACTAGTGAGAC» «^ CA pGTACCTC (^ CTGCAAGCmGAAAGAG ^ 5625 5626 ATATApGTGAGApGCACATGpATGTGAGp < »^ T 5700 5701 CAGTTCTCAAGAAGApa AAACTGGCTAGaCGACTAev ^ TGACa ^ W ^ 5775 5776 TATCCTGKTsCCOlAGGGCmACTAAGCs 5850 5551 GCTC GAGTGCCAGGGTAG AGAAAGaTGGTCACaCWGACAGaTACM 5925 5926 ATCACGACTCKTGAACTGTACG7GGAAAGGG «AAGT (^ TaGGTCD ^ 6000 6001 TOTCpCAAAAA8TmGAApA7GTTpCC7GAAAA7GAAA ^ 6075 6076 TGG7CGAGQA7 (^ ACTAG_7_GG <? CW7pATGGAAGAAACAGC7AACmGAA ^ 6150 6151 A7ATAAGGA7CTGACCGCG7G¡X7GCCAGM7G (_GA ^^ 6225 6376 AAAGGCA7ATGAATGGGAAAAAGCTGGCCG 7CCGATCG7GTACAAA7 (1AGGM 6450 6451 G6pATCACA (ÍAGCA7AaAACTAmAGC7CCA7A7CA ^^ 6525 6526 pG7CApaCACGA7GAAGTTmGpG7CpGATGGAMTM7AGCCAAAG7AmC7AGCC7A7CC7CA ^ 6600 6601 AC ^ 7G7 < ^ 7GA7GACAKTGTGTCAAAGTCA7CpATCCCA7GCG7GI 6675 6676 TAAAGC7AT7a7A7GAAAAM7CCpAßAGAAC ^ rpGp (XlAW7GCAAC 6750 6751 ApGTGCAATAAACCGGpGAIGGAAGTAGpCCACApAAGCATGAGCACICATT ^ 6825 6826 GG7AGVWlAAí? CAACAmAGTGA 7CCTCApCCTaACMTC ^ 6900 6901 GGGTACCWTa7AACCA7GCTAGCCATGAAC (TpcrrGGACAT ^^ 6975 6976 CfcTGGAMpcpGCpCTCpCAGAAACC ^^ 7050 7051 (LATGTCTAAGu ^ AAAGATGACCTi GAA ^ 7125 7126 AAGAAAAGA7 (XA (i4CTs87AGAAfiAíy ^^ 7200 7276 CTATATGACAGGAAAA (iAACTTCGCCA (^^ 7350 7351 CCGVKAAmsCCTGCCCAGGCATCsCCT ^^ 7425 I 7426 ATOTACAG7AG7AGA7C / apACTGCCGpttA ^ 7500 l 7726 mMT6 ^ GTOTAA GfcTmCTAaT7aTCCT ^ 7800 7801 AGCGCCAaG4A7 & £ 7GGAíAA-rr7TjrCMTGAA ^ 7875 7876 ACAAGffrG7AA7CAA < iAsCGAAATAGAGTGA < ^^ 7950 7951 ACAA6AAGC7AsGA7GAAAAmACTATGCDW ^^ 8025 8026 GTAAAAGAA7A7Gp T 7C7AAAACYTAATa? Tp8TATGAATCTCTGGpCTA7C ^ 8100 8101 TACAACATAAAmAapaCAGC? ACTpTATp CTCTGATCApMTTATATGTAA ^ 8175 6176 AGTOpAACiAAAIAAAaGCTpmMTAAAAAAAAAAAAAAAAAAAAAAAA ^ 8239 of Sequence No. 2: p rotate A na of ATR 1 MGEHGLELASMIPALReLGSATPEEYNTWQKPROILCQF 1 DR1 TDVKVVAVELVKKTOSQPTSV 66 67 f DFlQHIHKSSPuTVHVSGSHEfttGSCIEF ^^ 141 142 FGVLTKEtL FEDLVYLHRRNVi VEWPVVMSJ Li ^ 216 217 lAIVFFRROELLL QlIXVLLEYGSPKIKS ISFLTELJOLGGLPAOPASlFFSSFLELL HLVEMDTOQLKLY 291 292 EEPtS &tisiFPFEAEAYIWIEWlNrClEia.CWFeDGYLIffiL & 366 VIKVWI ^ ^ 367 441 517 ICALIDVIGIETOMYUGPLYAALKMES FWXQHKSI CKPSVVlT MS VLKSCI Fn + LLESVQKLOLEATIOKVVKlYOALIYMOVNSSFEOHILEOLC 591,592 (666,667 HSLPWI YSHSDOGCLKLpFAANLLILSCRISDSYSPQAQSRCVFLLlLFPRRl FLEWR7AVYNWALQSSHEV IRAS? SGFFILLQQQNSC? RVPKILIDKVK8SOIVICXE ^ 741 742 CRNLW7SQl «CSSS0LI (ASVC PFLFLLKKKPSPS LAFI»? HHLCKHLC> FREK 816. 817 DKDVRVAFSGNIKH1LESLDSEDGFI ELFVLRHKEAYTHAOISRNNELKDTL t LpGDIGRAAKGDLVPFAllH 891 B92 LUCllSKSASVSGAAnEIRAlVAAKSVKLQSFFSQYW? Pia ESL ^ 966 9670f £ r ^ NTLSEIAfftTDFPDIJ TR7lQVlLPDLAA) CA ^^ 1041 1042 CSCSi EIW? E7EIttGSllRQDFCaKr * lLlRIGEHY ^ U16 1117 The? QPiaLGILAFFNM (SSSVGIE_Xi NSLMSLI * ^ 1191 1192 CCTAWKFVrcLDr GSLLSHVIVALLPLIH 1266 1267 VLQEYI «ETSES7T Q aSNKAIQHENVDVRIHAL7SLKETLY | i-S, QEi iKYATDSE7VEPIISQLY7VLLK 1341; 13426C () 0A16OARLLCGE GeLGAI0PGRLDFSpE7O5KOFtFV7Gve? SSFAYGLLMEL7RAYLAYAONSRAQ0S 1416 1417 MYAIQELlilYÍ EKpNGPGi WRRFPEHVRElLEPHLNIRYKSSOKSTD '? GVICKPiYISKLGSNFAEWS 1491 1492 ASWAGYLl7lCVRHOLMKipcCSIt FFL? VllYLLPHILVYVLLGCN0CDs3EYYAE] R VL HD0 () H7IM71566 15670OIAS0LC0LS7QTVFSKL0H.7QWARHKF0ALKAEKCPHSKSNRN'KVDSHVSTVDYE0YQSV7RFLOL IPOOIL 1641 1642 1716 AVASFRSAYIRAVWFESF17EKKWI0EHLGFL0ÍLYAAWEP0GVAGVSAIRAEPSLKEQ 1717 M7ACYDRAI0iEP0QllHYHGVVfór GaSTVp ^ 1791 1792 AADG «7TS GQLLl ^ AKKraiTAp? SL LVRAEOIVPLSAASFEI ^ YORGYEYlVR. ^ 1866 1942 NAUJIAGESRL.tfLYVElWWlWSKGOVHOM.IVLQIGVELCi ^ 1016 2167 YFl? YIWMHffAVSlCSSI NCKEIL ^ 2241 2242 Hn jaVEEATreiLIPUJSVHIrTUSILGTI ^^ 2316 2317 SOtOTIMuPIOOUtJFCM ^^ 2391 2392 ll «EK (W GIEUIC« AALSElCLKrTR ^^ 2466 2542 UIFDQREPLHSVUCTFLIFPLVEI mnCGHSI? A 2616 2617 CGHVHYLIOEATKNUCam.G rPYH 2664 Sequence No. 3: rad3 sequence 1 GCTACCAßTAAAAAaGCpAG7AAG7A7AAAACACAGAA6AATCCK 75 76 TGACGC7CCA G? CGCTA7AG7WTGAGAAC7AGGATGTGu3ApATAACpATC7 ^ 150 151 TAmA AKiMTAApGMpAAAAsCATTTOTCTm 225 226 ACTACCTpCAACAA7AACp I1HIII IGmApGACCApAT TCACATCAAAACTCAAAAAApCAATCA 300 301 pA7 (> GAAA (a7CCAGCsMTATTACpAAAAGpA 375 376 ApAGWTCGsC6ATACpAGTGCACCA7GCATOT ^ 450 451 TGCCACTTTTCY ^ TMpaApcy ^ TA ApACpACAATCGTCTTpATAM ^ 525 526 CGCGtGTGT7GCGT AAAAAGGCCTTTTmGAApGVW ^ 600 601 AAAGGAAAa iOKlCGATCmWCCAGAGCpA ^ 675 676 AAG7ApAGCApA9 AAGAAW7CVfipAGGTAGAAGTM ^ 750 751 AAGTm TTGAAGTTa7CpsA_ £ TpTAAACA7 825 976 TAmGACT < * pAAAAGTTraTAAGCTMmATW 105C 1051 A7GCpCffmA7GCGpApGGM7ApATApACAAAa ^ 1125 1126 cApacpACAAGAia: cc7CAaxMpcCMCtt ^^ 1200 1201 AGG? CAMmGAmACpA7CAp 7A7AGAGGAGGCpG7ACAl ^ 1275 1276 (_ACAATGCTCA7AmAAAGAM7CC TTpCC7AGTCCACCTC7CTT7AW ^ 1350 1351 GTTTTmAATTTrepA7TpApAAMCGM7CAGW7CGGAGAC7 (CAGC7C77 ^ 1425 1426 G7A7AG7 (XAAACmA7Gs (rrtmCCpGAATTpA7 1500 1501 A7A77p lG7CC7CCATCAACp WpGGGATCApGAASAAAACpA7AWGpGC7AATAC7GCTA7A71575 1576 C7CrTTTT 7C7CmAmGTmACCCAAAC7GTA ^^ 1650 1651 mCTG pWA8ApAGA (K ^ Cp (lAACCA6ApaGA7C7 «AAA ^ 1725 1726 G * TACCAAAGpCAGAAA7AGA7C ^ CAAsC7 ^ 1800 1801 CGTTGTTpCTAACisGMpAAACCTTmCTTpAC ^ 1875 1876 pAAAG7 (^ CCAGAA7GGTa7A 7AIG7ACAmGCAAMI (3GTGTA7A CAACAAC7TA ^ 1950 1951 C (HCTMmAAAGCACmCGTGAM7GpAGApApA7AK7CTCA7M 2025 2026 TGAACWCpC7C AGTTpG7G7AC7CATGpGCAAAGGT (ÍM ^^ 2100 2101 A7GAAG7ACA7GGpG7GAAGTrTGTAAC7CAI I l i IAC7A7pGA7GAGCGG7CGCC7p7AAAATTCCp 2175 2176 A7CACGAApGTTpG7GCApGCTAAAAAA7CCCGACA ^^ 2250 2251 AT < TTTpCß.TGGAGCCAGCApGCTC 2325 2401 CTMTMTCpGATATAGp (MmCA (Mß6MAGTAAAGCLTrGA 2475 2476 T (XAAAATACAGCTAppA6rWßßTCATpmCCTGGATCT ^ 2SSD 2SS1 TACAtT 6TACTAp6? AAmATATCp (G TMOtíXGM 2625 2626 CTCTGCMC pGCCKGAaXGTC? TATATCúTTTGGCMpM 2700 2851 GTATA ^ GAA? TKA (^ AAGTGATGTl ^ 2325 2 6 p AGGAmta: tMtpGG * A & tí? S 3000 3001 ttGmAcmmGp £ Gtctm (? JAmaApAc twmmM 3075 3151 TGGCMTAACMpACmATGATTTppAATMTOMpmm 3225 3301 TATLmGAGGTGcmc cpG ^ pAo GTAmr AAm 3376 TAAßGTmATW ^ 7CAAAßCpGGpt ^ GTpGATApAGW 3450 3451 Gm t tGTMtmAct? XmAt? CpAm 3525 3526 tmtmAtp? PtcmcAaMMTGm? CAAm 3500 3601 CGT6mAGCCpMGGCTAAAGAMTATitTG GsTÜlAMTG ^ 3675 3676 TAp? GmA? AACG ATGACCA mGpATp tpO ^ 3750 3751 CCAAGl &SMWpAAKímCTAApffiM 3825 3826 TTp &TTi GTGTAAAATA? XpCMCAMtATGÜM 3900 3976 AeßGrWAGAAAepWfflTAmTM 4050 4051 ATCTAMGC? CArWpmpßCCTATGets < & WMTTKTA? AGm 4125 4126 p TAAAAAAAAGGGttTAACTGmTAAnGrWTIRGAtm 4200 4201 TACMTrmAACpCOMTATamAACACC? TKCQM 4275 4276 ATßpACTApCATACpGGATGCAGl MI 11 acnAMpGTfMpACGCCapC &? AAOXTGAM 4350 4351 AAATAmGGTATmpC &mTA $ TlMAGKCAAGAß6pAA & 4425 4426 TmAMTGpATmAAC (XATC KTGG pATAA C ^ 4500 4501 MC (XGGACCT (ÍA? GGApAAApCCGTGGGGÍM (MAGATACAaTCAT ^^ 4575 4576 T (stpACCpAACAAATGGCpC (^ J (AßA GGAATlT ^ 4650 4651 AGAACCGp? TATGKGGTGGMGATGCTACCmCGAGAATCApMTCTCAAAAG 4725 4726 AtmcpcA ACApAmAm ?? mAupGT6Amamt < xt BOQ 4801 AACAUTACGTM? GCTA (MCTCCATATG (M AGAGTCCGApATA6M 4875 4876 (Mn6ATGATC (MATGMT¡AGCATmpAMTpCaT ^ 4950 4951 TaTGAmpCAQ CTGG6mG6CmwpmACAApApAp (MA ^ 5025 5026 AAAA (&GAMTCGGm6CpMCAGCAtGCT6MTCGGG6apATGM ^ 5100 5101 CTmATmMTGACAACO £ 6AGTApCGAAGAT6pAAAmG &; 5175 5176 CTATT & TT (XnAAAAAAGTGTCm? ^ AAAGCAAT ^ 5250 5251 AOMTA (XTAa¡GAGGApcptmTMTWCGSC6? S 5325 6326 CAApGÜMACAGGCGCCCATTCAGCTAT6ATT ^ 5400 5401 p GGAT TL ^ C7GC AppTCCGA cpGATATlG 5475 5476 TA < XTCCGTACG6tmpCAAGUC (MATCCmCCAC7UCpAdpG ^ 5550 5551 AGAAMCTGCTGAAATATATCTCGAGApGCAAGrWAKKGAAAAM 5625 5626 caKCTCAAAGCAAia ^ rRAGATAM (XGCTA6CAACMTAGAßC ^ 5700 5701 AAaTCGTAAAGCT? MCTGAApGAATTmCGpAATAACMCATGT ^ 5775 5776 AAAGACCTAAHUTCGTAMGAAACmAGGAMTCWpAAAGGAAM $ 850 ' 5851 TCGGAAAAGCfGGC (MCT6GGApGAA6GAm6GA ^ 525 5926 GTGAGAATACGApApAT GGaTATCGAG t GTATW 6000 63766mßACmtG6CApAATüGC? UM ¿450 6976 GATpAT (^ TGGGTAMTCAT? CT ^^ 7050 7051 CttTAT TATC AGA TCA A (mGAm? G aTTBC ^ 7125 7126 * O MTOTA (XGAMm (XTCCAipTT ^^ 7200 7351 AGBTOTACTT? G AA C ^^ 7425 7426 CAGGpATGAAGQGGrmWfiGAAAí ^ AGQ ^ 7500 7501 TGAQCGTACTAGAC ^ m (rrACAT (-ATAMAGTttACTGtiAATAGA 7575 7576 ATAATGAAOJ ^ TGAAGTTTTGGATATAApca ^ AAAAATpCAAG ^ 7650 7651 TATaApG? AGGGCA pCAAGMTTWTCAMTaOTCTCAACCCAAAA 7725 '7951 ApGAAAAAAAGCTATA7CT < _ cTGAGT < * TCA ^^ 8022 In italics, sequenced by Seaton et al. In bold are the bases removed in Seaton et al. (2499, 22501, 2507, 2509) The two bases on both sides of a single insert are underlined C (5918/5919) in Seaton et al. (ie, the incorrect base is not shown, but if a residue is shown on both sides) Sequence ID No. 4: rad3 protein 1 MSOHAKIttAGSLD PRGlMPWGtXl ^^ 75 151 FLVaYA5FMRYWKYYITN \ "IVL0i 7FPYKMrePNSQPL05ISPNYPTHRED FDLLlINI ^ 225 226 SAFFFAQCSYLI TONFPSPPFTAlMIKPsFi YILl ^ 300 301 CeKSKHIUiSSINLTl &LKlcmVANTAISLFFLSLFVl ^ 375 376 ILFKCRYQSSElD RAF6EICTaLE.m.FSN $ ElNLFlL! MSÜ 450 451 NNLJXPSr? HFREri.DYYS HNV7ISEDIMNFSLVL ^ $ 525,526 600,601 PFKIPYHELFCALLKNPDl ISSSVKQSLLLCGFFRWSQHCSNFNKESMISLREF1KKALAS7SRCLRVVAAKVLP IFIKGPNNLDlVEFHKESKALlFNlLKl VENTAILETVILSWISLSftVVEEEELHFVLLEVlSSVlNSGirYO 675 676 GlGl ^ lQQIAS7 HIS LSPYW VSVA] V (X3 ^^ 750 751 MLmiAELSQSDVATLCLTt? MILASLipiWMeSmLLSl? TSDFeKmLm 825 826 K ?? IDIW £ ÍC / £! K ^ 900 901 em. ltm Gim.mQSAFM [HLmAlrWSLlUmPEYSSlA l Ln 975! 976 L? IQlFDFlSSDmCLKLKMlPTSWSJWS? WlFCSlQrW 1050 1651 JtrefflOTAf / rifWWS »^ 1725 1801 ílPVKQS- VTRIIfcF ^ lY? GTWIYESH ^ 1875 2026 DITlAK0AWKWPFI > 7 (FRl> raeV0IW ^ 2100 2101 lai-JWXANRi ^ CIRTm.PLt € ^ 2175 2251 i F0KaT7FJ e? Vf 7r * WIGPGrc 2325 2326 SSK t # ieAf £ VU) I.imQGFN > Gm 2386

Claims (17)

1. REI INDICATIONS 1. A polyp leotted in a substantially isolated form capable of selectively hybridizing with sequence ID No. 1 or its co-elements.
2. 2. A polynucleotide according to claim 1 comprising sequence ID No. 1 or a fragment of my SYMBOL.
3. 3. A polynucleotide probe comprising a fragment of at least 15 nuclei. of a pol i nucleotide in accordance with that defined in claim 1 or 2.
4. 4. A pol lyotylated leotard in isolated form comprising sequence ID No. 3 or its complement.
5. 5. A pal i nue 3 eot gone in a substantially isolated form comprising cps. of the sequences set forth in sequence ID Nos. 2 or 4, a polypeptide substantially homologous thereto, or a fragment of the ID polypeptide. of sequence No. 2.
6. 6. A polynucleotide in substantially isolated form that codes for f. The invention is in accordance with claim 5.
7. 7. A vector carrying a core 11 in accordance with claim 1, 2 or 6.
8. 8. An anti-focal point for the poly. ept gone from sequence ID No. 7 or fragments thereof.
9. 9. A method to determine the presence or absence of a polynucleotide in the form defined in Relation 1 in a sample of human or animal body, which is set: put a. sample of corpium humartci or animal that c n erμ DNA or APN in contact. on a probe comprising a polynucleotide or primer d < - conformity with that defined in claim 1 under hybridization conditions; and detect any formed between the probe and the nucleic acid in the sample.
10. 10. A method for detecting polypeptides in accordance with that defined in claim 6 present in biological samples, which is e.g. (a) providing an antibody according to claim 7; (b) incubating a biological sample with said antibody under conditions that allow the formation of an antigen-antibody complex; and < c) determining if an antibody-antigen complex comprising said antibody is formed.
11. 11. A test method for screening candidate substances for cancer therapy, comprising: (a) providing a polypeptide of the invention that preserves the lipidkinase activity and a substrate for said qumasa under conditions and cort reactive such that the kinase activity acts in the substrate; s (b) putting said polypeptide and substrate in contact with a candidate substance; (c) measuring the degree of distribution of the qui asa activity of the polypeptide; and (d) selecting a candidate substance that provides a distribution of the activity.
12. 12. A test method for screening candidate substances for cancer therapy, comprising: (a) (i) incubating a polypeptide of the invention cut another as a polypeptide of the invention, which may be e) same as the first polypeptide or well different from the first polp, or conditions that allow the first polypeptide to be linked to the second polypeptide to form a complex; (i i) placing the complex formed in this manner in contact with a candidate substance; or (a) incubating a palpeptide of the invention with another polypeptide of the invention, which may be the same as the first polypeptide or different from the first polypeptide, under conditions that allow the first polypeptide to be linked to the second polypeptide for form a complex and in the presence of a candidate substance; and < b ^ determine whether the candidate substance inhibits the binding of the first polypeptide to the second polypeptide and (c) select the candidate substance that inhibits the binding s of the first polypeptide on the second polypeptide.
13. 13. A method according to claim 12, wherein said first polypeptide can be ingested from said second polypeptide.
14. 14. A method for the treatment of cancer in a patient, comprising administering to said patient a therapeutically effective amount of a candidate substance sold according to the method of any of claims 11 to 13.
15. 15. A method to increase in a patient the susceptibility of cancer cells to chemotherapy and / or radiotherapy, which comprises administering to said patient a therapeutically effective amount of a candidate substance selected in accordance with the method of any of claims 11 to 13.
16. 16, The use of a candidate substance selected in accordance with the method of any of the claims 11 to 13 for the treatment of cancer.
17. 17. The use of a candidate substance selected in accordance with the method of any of claims 11 to 13 to increase the susceptibility of cancer cells to chemotherapy and / or radiotherapy.