WO1998042869A1 - Cribles d'adn primase de mammifere et agents modulateurs d'activite - Google Patents

Cribles d'adn primase de mammifere et agents modulateurs d'activite Download PDF

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Publication number
WO1998042869A1
WO1998042869A1 PCT/US1997/004654 US9704654W WO9842869A1 WO 1998042869 A1 WO1998042869 A1 WO 1998042869A1 US 9704654 W US9704654 W US 9704654W WO 9842869 A1 WO9842869 A1 WO 9842869A1
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Prior art keywords
primase
dna
activity
agent
enzyme
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PCT/US1997/004654
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English (en)
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Michael Kozlowski
Junko Aimi
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Geron Corporation
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Priority to PCT/US1997/004654 priority Critical patent/WO1998042869A1/fr
Publication of WO1998042869A1 publication Critical patent/WO1998042869A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1235—Diphosphotransferases (2.7.6)

Definitions

  • the invention relates to methods for identifying agents that modulate the catalytic activity and/or stability of mammalian DNA primase, compositions of such agents, improved DNA primase assay methods suitable for high-throughput screening to identify such agents, and the use of such methods to identify pharmaceutical agents and laboratory reagents which modify DNA primase activity.
  • CROSS REFERENCE TO RELATED APPLICATIONS
  • DNA primase initiates DNA replication by the synthesis of ribonucleotide primers which serve as a substrate for elongation by DNA polymerase activity.
  • the primase activity synthesizes short oligoribonucleotides during initiation of DNA replication and elongation of the lagging strand.
  • DNA polymerase elongates the RNA primer to complete the synthesis of the Okazaki fragment (Stillman B (1989) Ann. Rev. Cell. Biol. 5: 197).
  • Okazaki fragments are then extended by either DNA polymerase ⁇ or e, allowing DNA polymerase and DNA primase to recycle and initiate another Okazaki fragment on the lagging strand ( aga S and Stillman B (1994) Nature 369: 207).
  • This essential role of DNA primase makes it a key component in the regulation of initiation of DNA replication.
  • DNA primase (as well as RNA polymerase) is the ability to synthesize oligonucleotides de novo on a template by the formation of an initial dinucleotide.
  • DNA primase initiates synthesis with a triphosphate purine moiety at the 5 ' end (Gronostajski et al. (1984) J. Biol. Chem. 259: 9479).
  • the primer template is translocated intramolecularly to the active site of the DNA polymerase subunit.
  • Mammalian DNA primase and DNA polymerase copurify as a complex containing four subunits with approximate molecular masses of 180, 68, 58, and 49 kD.
  • the 58 kD (p58) and 49 kD (p49) subunits can be separated from the 180 kD (pi 80) and 68 kD (p68) subunits and retain DNA primase activity when present as a p49-p58 complex.
  • the p49 and p58 polypeptides are conventionally referred to in the art as the small subunit and large subunit of DNA primase, respectively.
  • the large and small subunits of DNA primase have been cloned as cDNA from mouse (Prussak et al. (1989) J. Biol. Chem. 264: 4957; Stadlbauer et al. (1994) Eur. J. Biochem.
  • DNA replication inhibitory agents are used as human and veterinary drugs, such as antineoplastic agents (e.g., arabinosylcytosine. thioguanine, 5-fluorouracil, hydroxyurea, mitomycin, daunorubicin, doxyrubicin, actinomycin D, cyclophospha ide, etc.), antiviral agents (e.g. , AZT, 3TC, ddl. acyclovir. gancyciovir, foscamet, etc.), antifungal agents, and the like. Identification and development of new modulators of DNA replication and/or DNA repair synthesis provide new. improved, and/or alternative pharmaceuticals to treat diseases such as cancer, viral diseases (e.g.. hepatitis B. AIDS), and other pathological conditions.
  • antineoplastic agents e.g., arabinosylcytosine. thioguanine, 5-fluorouracil, hydroxyurea, mitomycin, daunorubic
  • DNA primase modulators could serve as candidate pharmaceutical agents to treat a variety of diseases, as well as laboratory reagents, for example as controls in a screen for primase activity, or for agents that modulate primase activity. Because of the large number of potential agents which can be screened for the activity of modulating DNA primase activity. it would be desirable if such a method were suitable for high-throughput screening of compound libraries. It would also be desirable to obtain compositions of specific DNA primase modulators, particularly agents which selectively modify DNA primase activity as compared to any effects on other enzymes involved in DNA metabolism (e.g. , DNA polymerase ( ⁇ , ⁇ , y, ⁇ , e), helicase or telomerase) and/or RNA metabolism (e.g.
  • DNA polymerase ⁇ , ⁇ , y, ⁇ , e
  • helicase or telomerase e.g.
  • RNA polymerase (pol I, pol II. pol III) and related proteins).
  • RNA polymerase (pol I, pol II. pol III) and related proteins.
  • DNA synthesis/replication inhibitors are nucleotides, nucleosides. and analogs thereof, and much scrutiny is directed at developing this class of agents, it would be desirable to have a method suitable for identifying DNA primase modifiers which are compounds other than, or in addition to nucleotides or nucleosides.
  • the present invention fulfills these and other needs, and provides methods which will find wide applicability in the art.
  • the present invention provides new methods for identifying agents that modulate mammalian DNA primase activity.
  • mammalian DNA primases such as human DNA primases
  • the method can be suitably adapted by those skilled in the art for identifying modifiers of non-human (e.g. , murine) DNA primases as well.
  • the method can be practiced with reference to DNA primase activity associated with DNA polymerase ⁇ , primase activity associated with other DNA polymerases, or primase activity associated with any mammalian telomerase.
  • Such adaptations will be apparent to those skilled in the art in view of the present disclosure of the general method and specific embodiments provided.
  • the present invention provides compositions and methods for screening for agents which are modulators of one or more functions of mammalian DNA primase or its activity and agents which can modulate DNA primase-mediated cell replication and/or modulate neopiastic and immune conditions, as well as other pathological conditions dependent upon DNA primase function or activity.
  • the invention provides methods for identifying agents which modulate a mammalian DNA primase activity.
  • An example of primase activity, or function. is the synthesis of a primer, optionally followed by polymerase elongation of the primer to create a complement to a template nucleic acid.
  • a second example of primase activity is the direct or indirect binding of primase to a second molecule, such as a polymerase, an antibody, or the like.
  • a composition comprising a mammalian DNA primase enzyme is provided. The enzyme is optionally from a purified or partially purified natural source, or is optionally a recombinantly produced enzyme.
  • the primase enzyme optionally comprises a moiety which is unrelated to a native primase enzyme; one such enzyme is a recombinant fusion protein comprising a primase domain and a second domain (e.g. , in a two-hybrid system).
  • Example compositions comprising the primase enzyme include in vitro aqueous reaction mixtures, cells and organisms.
  • the composition is contacted with the agent and the activity of the primase enzyme in the presence of the agent is monitored.
  • This agent is optionally added to an in vitro reaction mixture, or a cell or the like which comprises the primase enzyme, wherein the activity in the presence of the agent is an indicator for whether the agent modulates primase activity.
  • the activity of the primase enzyme is monitored by measuring incorporation of a nucleotide label into a nucleic acid polymer, for which primase initiated or primed synthesis.
  • the activity of the primase enzyme can be monitored by measuring the binding of the enzyme to a polymerase protein or substrate.
  • the methods of the invention can include a control reaction in which the activity of the primase enzyme in the absence of the agent (or the presence of a control agent which modulates activity in a known way) is monitored and compared to the activity of the primase enzyme in the presence of the agent.
  • a control reaction can be omitted, as for example, when a large preparation of primase with a known activity is available.
  • Multiple assays are preferably performed in parallel in the methods of the invention, with several agents and/or several activities being screened simultaneously, a decided advantage if large numbers of the agents are to be screened.
  • Nucleic acids produced as a result of primase activity include DNA and RNA polymers. These polymers can be produced by the action of primase alone, or by the combined action of primase and a polymerase such as polymerase . The production of a nucleic acid as a result of primase activity provides a measure of primase function for the primase enzyme. Nucleic acid production is typically measured by monitoring incorporation of labeled nucleic acids into the nucleic acid polymer, or by hybridizing a probe to the polymer, or by measuring total DNA or the amount of duplexed or single stranded products.
  • a probe is hybridized to a primase reaction product, with the amount of probe bound providing a measure of activity for the primase enzyme.
  • the probe or product is immobilized or captured on a solid surface, which is optionally washed to remove non-specifically bound components after hybridization with primase reaction products or probes to the products.
  • the assay includes a blocking agent, such as albumin, a nonfat milk protein, polyvinyl pyrrolidone, or Ficoll.
  • This invention further provides methods of modulating the activity of a mammalian primase.
  • an agent identified using an assay of the invention is contacted to a composition which includes the primase molecule.
  • the agent then potentiates or inhibits the activity of the primase enzyme.
  • Example inhibitors include Primase modulators 1-25, the structure of which are shown in Figure 1.
  • Examples of Preferred modulators include primase modulators 17. 19 and 20.
  • compositions include agents identified using an assay of the invention. These agents are identified, for example, when they are purified from mixtures used as agents in the screening assays of the invention, including combinatorial chemical libraries, cellular extracts and the like.
  • the compositions comprises a primase molecule.
  • the invention provides a composition in which an agent is bound, permanently or transiently, to the primase molecule which the agent modulates.
  • the invention provides methods for identifying DNA primase modulators or modifiers, such as a DNA primase inhibitor or DNA primase activator or potentiator.
  • the method identifies DNA primase modifiers which produce: (1) a detectable alteration in DNA primase activity, such as the capacity of a DNA primase to initiate oligoribonucleotide primer synthesis and/or the rate of chain elongation of a nascent oligoribonucleotide primer catalyzed by DNA primase. either alone or in conjunction with a detectable alteration in DNA primase activity, such as the capacity of a DNA primase to initiate oligoribonucleotide primer synthesis and/or the rate of chain elongation of a nascent oligoribonucleotide primer catalyzed by DNA primase. either alone or in conjunction with a
  • DNA polymerase e.g.. DNA polymerase
  • DNA primase/DNA polymerase complex e.g., DNA primase functionally bound to DNA polymerase a
  • dNTPs deoxyribonucleotides
  • DNA primase modifiers can alter the catalytic activity of DNA primase and/or the binding of DNA primase to at least one predetermined binding partner (e.g., DNA polymerase ⁇ ).
  • the method comprises identifying as candidate DNA primase modifiers those agents which modulate primase activity.
  • the modifier is added to a DNA primase reaction comprising: (1) an enzymatically active DNA primase, (2) a suitable primase template polynucleotide, and (3) a primase reaction mixture containing at least one species of labeled nucleotide capable of being incorporated into a product polynucleotide chain (e.g., such as a dinucleotide or longer polynucleotide) by the catalytic activity of DNA primase, result in a detectable and reproducible increase or decrease in the amount of product polynucleotide produced in the reaction as compared to a standard or control reaction which is substantially identical except which lacks an added agent.
  • a DNA primase reaction comprising: (1) an enzymatically active DNA primase, (2) a suitable primase template polynucleotide, and (3) a primase reaction mixture containing at least one species
  • the DNA primase is a human or mouse DNA primase composed of large and small subunits obtained by expression of an encoding recombinant polynucleotide expression construct.
  • the DNA primase subunit(s) can be expressed in prokaryotic or eukaryotic expression systems.
  • the primase template polynucleotide is a single-stranded DNA molecule; in a variation, the ssDNA template comprises the sequence 5'-GCTTTCTTC-3' or 5'-GCTTTCTTCC-3 ⁇
  • the primase template polynucleotide is a double-stranded DNA molecule having a portion which is non-complementary or looped out to form an open helix replication bubble suitable to serve as an initiation locus for DNA primase.
  • the labeled nucleotide is a biotinylated, fluorescently labeled, or radiolabeled ribonucleotide, such as can be made using appropriately labeled nucleoside triphosphate
  • NTP e.g, ATP, GTP. CTP. UTP, TTP. ITP. or the like.
  • one or more labeled deoxyribonucleotide species can be used; such as a biotinylated, fluorescently labeled, or radiolabeled deoxyribonucleotide, such as dATP.
  • dGTP, dCTP, dTTP, dITP or the like.
  • the amount of product polynucleotide is determined by contacting the reaction mixture, following a suitable incubation period, with a substrate which selectively immobilizes or binds to polynucleotides and which substantially does not immobilize or bind to mononucleotides or labelling reagents:
  • a substrate which selectively immobilizes or binds to polynucleotides and which substantially does not immobilize or bind to mononucleotides or labelling reagents:
  • a substrate is a charged membrane (e.g.. a glass fiber filter such as the 2SC fiiter from Whatman, Nylon 66. nitrocellulose, DEAE paper or the like).
  • reaction products can be chromatographed or electrophoresed (e.g., PAGE) to separate polynucleotide products from unincorporated nucleotides or other materials.
  • the reaction further comprises a DNA polymerase activity, typically a mammalian DNA polymerase a, and reaction conditions suitable for catalytic activity of the DNA polymerase(s), such that product polynucleotides formed by the activity of the DNA primase may be extended further by the DNA polymerase, which can be useful to enhance or amplify the signal resulting from incorporation of labeled nucleotide into product polynucleotide.
  • one or more labeled dNTPs are also present in the reaction mixture, typically including each dNTP which would be present in a complementary strand of a template polynucleotide.
  • only one species of dNTP is labeled (e.g...
  • the method employs a DNA primase and/or DNA primase/DNA polymerase reaction comprising: (1) a template polynucleotide capable of providing a template for a mammalian DNA primase; (2) a labeled nucleotide or polynucleotide species, and optionally (for heteronucleotide template sequences) unlabeled nucleotide species such that the reaction contains nucleotide species (either labeled and unlabeled) representing nucleotides which can be efficiently incorporated in a complementary strand to the template polynucleotide; (3) a predetermined amount of mammalian, preferably human.
  • DNA primase and. optionally, a predetermined amount of a mammalian, preferably human, DNA polymerase (e.g. , DNA pol a) in suitable reaction conditions (e.g. pH. ionic strength. ATP, temperature, metal ion concentration, etc.).
  • a mammalian, preferably human, DNA polymerase e.g. , DNA pol a
  • suitable reaction conditions e.g. pH. ionic strength. ATP, temperature, metal ion concentration, etc.
  • the DNA primase reaction typically contains either: (1) at least one labeled ribonucleotide species, and optionally additional unlabeled ribonucleotide species necessary for synthesis of a complementary RNA strand to the template, for direct reporting of RNA synthesis as a measure of primase activity, or (2) at least one labeled deoxyribonucleotide species, and optionally additional unlabeled ribonucleotide species and deoxyribonucleotide species necessary for synthesis of a complementary strand to the template, for reporting of DNA synthesis which indirectly reports primase activity as RNA primer synthesis is necessary for initiation of DNA synthesis.
  • reaction times can be shortened to reduce the average length of DNA chains synthesized and/or a DNA chain terminator nucleotide (such as. e.g.. dideoxynucleotide: ddCTP, ddTTP, ddGTP. ddATP, ddlTP, etc.) can be included in sufficient amount to reduce average DNA chain length which may be preferable to ensure o that DNA synthesis is substantially proportional to RNA primer synthesis (and that the readout, whether by labeled RNA synthesis or labeled DNA synthesis, is representative or proportional to DNA primase activity in the reaction).
  • a DNA chain terminator nucleotide such as. e.g.. dideoxynucleotide: ddCTP, ddTTP, ddGTP. ddATP, ddlTP, etc.
  • the product polynucleotide(s) generated by DNA primase and/or DNA polymerase activity(ies) are immobilized and detected by hybridization of a labeled complementary probe which specifically hybridizes to the product polynucleotide(s) and substantially does not hybridize to the template polynucleotide(s).
  • the immobilized polynucleotides are bound to a solid substrate (e.g., Nylon 66, nitrocellulose, etc.), and may optionally be blocked to prevent non-specific binding (such as with a pre- hybridization solution as described supra, and/or washed to remove non-specifically bound probe.
  • labeled nucleotides bear distinct labels to distinguish template versus non-template directed polymerization in a DNA primase reaction or coupled DNA primase/DNA polymerase reaction.
  • a first labeled nucleotide species having a first label is incorporated in polynucleotides produced from template-directed polynucleotide synthesis, such as DNA primase-catalyzed oligoribonucleotide primer synthesis or DNA primase/DNA polymerase-catalyzed elongation of a oligoribonucleotide primer by template-directed polymerization.
  • a second labeled nucleotide species having a second label which can be distinguished or discriminated (i.e..
  • a "nucleotide deficient template” serves as a primase template, and is a homopolymer or a heteropolymer polynucleotide composed of residues of two or three deoxyribonucleotide species (i.e., the template lacks at least one dNTP species) wherein at least one of said deoxyribonucleotide residues is a complement nucleotide of the first labeled nucleotide, and wherein none of said deoxyribonucleotide residues is a complement nucleotide of said second labeled nucleotide, whereby template- directed polynucleotide synthesis by DNA primase or DNA primase/DNA polymerase yields a product polynucleotide comprising an incorporated (i.e., poly
  • the second labeled polynucleotide species is complementary to a dNTP species which is not present in the nucleotide-deficient template, and therefore polynucleotide products of the reaction having incorporated second labeled nucleotide residues substantially represent reaction products generated by untemplated polymerization.
  • the method employs a DNA primase and/or DNA primase/DNA polymerase reaction comprising: (1) a nucleotide-deficient template and substantially lacking other template species; (2) a first labeled nucleotide species and a second labeled nucleotide species, and optionally unlabeled nucleotide species such that the reaction contains nucleotide species
  • DNA polymerase e.g., DNA pol a
  • suitable reaction conditions e.g., pH, ionic strength, ATP, temperature, metal ion concentration, etc.
  • DNA primase reaction typically contains a first and second labeled nucleotide species which are either both ribonucleotides or are both deoxyribonucleotides.
  • reaction times can be shortened, as described supra, to reduce the average length of DNA chains synthesized and/or a DNA chain terminator nucleotide (dideoxynucleotide: ddCTP. ddTTP. ddGTP.
  • ddATP ddlTP, etc.
  • the product polynucleotide(s) can be detected by hybridization with a complementary strand probe polynucelotide which may be labeled and/or which may be immobilized and used to capture, by hybridization, a labeled product polynucelotide having sufficient complementarity to hybridize under suitable hybridization conditions.
  • a labeled ribonucleotide species having a first label and a deoxyribonucleotide species labeled with a differentiable label are used to separately report DNA primase activity as labeled RNA and DNA polymerase activity as labeled DNA.
  • the DNA primase reaction comprises: (1) a template polynucleotide suitable for templating DNA primase activity; (2) a labeled ribonucleotide species comprising a first label (e.g..
  • 32P- ⁇ -CTP 32P- ⁇ -CTP
  • ribonucleotide species either unlabeled or labeled with said first label, necessary for efficient synthesis of complementary strand RNA primers
  • a labeled deoxyribonucleotide species comprising a second label (e.g.. biotinylated dTTP), and optionally other deoxynucleotide species (dNTPs), either unlabeled or labeled with said second label, as necessary for efficient synthesis of complementary strand DNA sequences to the template
  • a mammalian preferably human.
  • DNA primase and a mammalian, preferably human, DNA polymerase e.g., DNA pol a
  • suitable reaction conditions e.g., pH, ionic strength, ATP, temperature, metal ion concentration, etc.
  • RNA primer synthesis (DNA primase activity) and detection of the amount or relative quantity of the second label incorporated into polynucleotides reports DNA synthesis (DNA polymerase activity), while the combined information can be useful in detecting various types of inhibitors or activators, as well as in other applications as described below.
  • DNA primase activity DNA primase activity
  • DNA polymerase activity DNA polymerase activity
  • Each of the various embodiments of a DNA primase or a DNA primase/DNA polymerase reaction described can be used to detect and/or quantitate DNA primase activity and/or DNA polymerase activity and/or coupled DNA primase/DNA polymerase activity in a sample.
  • detection or quantitation of DNA primase and/or DNA polymerase activity has a variety of applications, including quality control assays for biopharmaceuticals and reagents (e.g.
  • the method of the invention employs a DNA primase or a DNA primase/DNA polymerase reaction which includes a predetermined amount and/or a predetermined activity of a mammalian, preferably human. DNA primase and.
  • the method is used to identify DNA primase modulators and/or DNA polymerase modulators from a library, or bank, of agents, or to detect such a
  • DNA primase modulator or DNA polymerase modulator in a quality control assay of a sample.
  • a DNA primase modulator when added in an effective concentration to a DNA primase reaction or a DNA primase/DNA polymerase reaction, produces a statistically significant increase or decrease in the amount of product polynucleotide (DNA, RNA, or both) produced in the reaction as compared to a standard or control reaction which is substantially identical except which lacks an added agent such as a DNA primase modulator.
  • a preferred measure of statistical significance is two standard deviations from the mean, wherein an agent which, when added at a maximally effective concentration or amount, produces a mean increase or decrease in the amount or rate or formation of reaction product polynucleotide which is at least two standard deviations outside of the mean of a set of control reactions which are substantially identical except lacking the agent.
  • Active agents which reduce DNA primase activity are thereby identified and are termed herein as DNA primase inhibitors.
  • Active agents which increase DNA primase activity are thereby identified and are termed DNA primase potentiators.
  • RNA primer synthesis is reported separately from RNA primer synthesis in the DNA primase/DNA polymerase reactions, it is possible to employ the method to identify agents which selectively or preferentially inhibit or potentiate DNA primase activity as compared to their effect, if any, on DNA polymerase activity.
  • an agent which significantly inhibits DNA primase activity as reported by, for example, RNA primer synthesis e.g..
  • RNA primers as reported by incorporation of labeled ribonucleotide into polynucleotide but which has substantially less capacity to inhibit DNA polymerase- cataiyzed elongation of DNA chains from the RNA primers (e.g., as reported by incorporation of labeled deoxyribonucleotide into polynucleotide) relative to the amount of RNA primer formation, is thereby identified as a selective DNA primase inhibitor.
  • an agent which significantly increases DNA primase activity as reported by RNA primer synthesis e.g.. as reported by incorporation of labeled ribonucleotide into polynucleotide
  • an agent which has substantially less capacity to increase DNA polymerase- catalyzed elongation of DNA chains from the RNA primers e.g., as reported by incorporation of labeled deoxyribonucleotide into polynucleotide
  • DNA primase inhibitors find use as commercial reagents and pharmaceutical agents.
  • DNA primase inhibitors can be sold and used for inhibiting or controlling DNA replication in mammalian cells, for example, cultured cells in a bioreactor producing a desired bioproduct for industrial or pharmaceutical use.
  • DNA primase inhibitors can be sold and used in quality control assays to determine the amount of DNA primase in a sample by titration of primase activity with predetermined amounts of the DNA primase inhibitor.
  • selective DNA primase inhibitors can be sold and used in to quench undesired DNA primase activity in a commercial product, such as an enzyme or polynucleotide preparation (e.g.,
  • DNA primase inhibitors also find use as products which can be manufactured and sold to research laboratories, similar to commercial restriction enzymes and other polynucleotide modifying enzymes which comprise a substantial portion of business activity in the "biotechnology" industry.
  • the DNA primase inhibitors can be used to inhibit mammalian DNA replication in mammalian cells, which is a desired property for certain procedures and experimental protocols; including control of undesired DNA replication in neoplastic cell types, types of virally-infected (e.g., EBV) mammalian cells, hyperplastic conditions, and the like.
  • DNA primase inhibitors are also suitable for use as pharmaceutical agents for inhibiting DNA replication in human pathological conditions, such as neoplasia, hyperplasia, viral infections, and related conditions.
  • DNA primase potentiators find uses as products as described for inhibitors, supra, but of course potentiate rather than inhibit DNA primase activity. In fact, this is a preferred aspect of the invention.
  • DNA primase potentiators can be used as pharmaceutical agents or otherwise for enhancing cell proliferation and DNA synthesis in cells, such as cells deficient in primase activity, such as can be the case in certain senescent cell types or conditions.
  • the present invention provides a method for identifying agents which modulate (i.e.. potentiate or inhibit) the intermolecuiar association of mammalian DNA primase and an associated DNA polymerase, typically DNA pol .
  • the method involves determining the capacity of an agent to alter the binding between a mammalian DNA primase and a mammalian DNA polymerase in suitable binding conditions.
  • at least one of the two binding species (DNA primase and a
  • DNA polymerase are labeled, and the other binding species is immobilized or provides a basis for immobilization of a bound complex comprising the labeled binding species.
  • a labeled binding species DNA primase or DNA polymerase
  • an unlabeled binding species DNA polymerase or DNA primase, respectively
  • the amount of bound labeled complex is determined as the amount of label bound to the surface.
  • a washing step is performed to separate unbound labeled binding species from bound labeled complexes immobilized on the surface.
  • the physical interaction of the bound labeled complex with the surface is reported, such as where the surface is a fluor or scintillant and the label in the bound labeled complex emits radiation suitable for activating the fluor or scintillant of the surface; light emitted from the surface reports the relative amount of bound labeled complex.
  • the DNA primase is labeled with a first fluor which absorbs radiation (particle or wave) and emits phosphorescent or fluorescent light at a first wavelength
  • the DNA polymerase is labeled with a second fluor which absorbs radiation at said first wavelength and thereby emits fluorescent or phosphorescent radiation at a second wavelength.
  • the labeled DNA primase and DNA polymerase are incubated under suitable binding conditions, and at suitable reactant concentrations whereby the amount of radiation of the second wavelength is approximately proportional to the amount of bound primase/polymerase complexes, and excited with radiation of the first wavelength (or particle type) and the amount of emitted radiation of the second wavelength is detected.
  • the relative amount of radiation of the second wavelength reports the relative amount of bound DNA primase/DNA polymerase complexes.
  • Agents are evaluated to determine their capacity to modulate (i.e.. inhibit or potentiate) the intermolecular binding of primase and polymerase in comparison to a control reaction lacking the agent.
  • Active agents can also be tested for their capacity to inhibit DNA primase/DNA polymerase activity in a coupled activity assay as described herein.
  • the present invention provides a composition comprising a substantially pure protein complex comprising a mammalian DNA primase polypeptide and a mammalian DNA polymerase polypeptide in binding conditions wherein the DNA primase is labeled and the DNA polymerase is immobilized, or wherein the DNA polymerase is labeled and the DNA primase is immobilized.
  • the invention also provides fragments of mammalian DNA primase and mammalian DNA polymerase which retain the ability to bind and form a primase:polymerase complex under physiological or test conditions.
  • the invention provides screening assays for identifying agents which modulate (e.g., potentiate or inhibit) binding of a human DNA primase polypeptide to a human DNA polymerase polypeptide and/or which modulate (e.g., potentiate or inhibit) binding of a mammalian DNA primase polypeptide to an alternative DNA polymerase- related polypeptide (e.g., human telomerase).
  • agents which modulate (e.g., potentiate or inhibit) binding of a human DNA primase polypeptide to a human DNA polymerase polypeptide and/or which modulate (e.g., potentiate or inhibit) binding of a mammalian DNA primase polypeptide to an alternative DNA polymerase- related polypeptide (e.g., human telomerase).
  • candidate therapeutic agents are identified by their ability to block the binding of a DNA primase polypeptide to a DNA polymerase polypeptide under binding conditions.
  • Compositions for identifying candidate therapeutic agents typically comprise: (1) a mammalian DNA primase polypeptide capable of binding to a mammalian
  • DNA polymerase polypeptide e.g., DNA pol a
  • DNA polymerase polypeptide e.g., DNA pol a
  • a DNA polymerase which interacts with DNA primase e.g.. DNA pol
  • aqueous binding conditions e.g., physiological conditions
  • a host cell e.g. , a yeast cell, mammalian cell, bacterial cell
  • a reporter polynucleotide or labeled NTP e.g., a medium to support growth or maintenance of a host cell; an agent is typically added to such a composition for evaluation.
  • a candidate therapeutic agent is identified by its ability to block the binding of a mammalian DNA primase fusion polypeptide to a DNA polymerase fusion polypeptide in a yeast two-hybrid system, wherein the primase fusion polypeptide comprises a primase polypeptide sequence fused to a GAL4 DNA-binding domain vector (GAL4 DB) or a GAL4 activation domain vector (GAL4 AD) and wherein the polymerase fusion polypeptide comprises a polymerase polypeptide sequence fused to a GAL4 activation domain vector (GAL4 AD) or a GAL4 DNA-binding domain vector (GAL4 DB), respectively.
  • GAL4 DNA-binding domain vector GAL4 DNA-binding domain vector
  • GAL4 AD GAL4 activation domain vector
  • a candidate therapeutic agent is identified by its ability to block the binding of a primase fusion polypeptide to a polymerase fusion polypeptide in a yeast two-hybrid system, wherein the primase fusion polypeptide comprises a primase polypeptide sequence fused to a GAL4 DNA-binding domain vector (GAL4 DB) or a GAL4 activation domain vector (GAL4 AD) and wherein the polymerase fusion polypeptide comprises a polymerase polypeptide sequence fused to a GAL4 activation domain vector (GAL4 AD) or a GAL4 DNA-binding domain vector (GAL4 DB), respectively.
  • GAL4 DNA-binding domain vector GAL4 DNA-binding domain vector
  • GAL4 AD GAL4 activation domain vector
  • GAL4 AD GAL4 DNA-binding domain vector
  • the invention also provides methods for identifying polypeptide sequences which bind to a DNA primase polypeptide.
  • a yeast two-hybrid screening system can be used for identifying polypeptide sequences that bind to DNA primase.
  • Yeast two-hybrid systems wherein one GAL4 fusion protein comprises a DNA primase polypeptide sequence, typically a full-length of near full-length human DNA primase polypeptide sequence, and the other GAL4 fusion protein comprises a cDNA library member can be used to identify cDNAs encoding proteins which interact with the DNA primase polypeptide, can be screened according to the general method of Chien et al. (1991)
  • an E. coli/BCCP interactive screening system (Germino et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 933; Guarente L (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 1639) can be used to identify interacting protein sequences.
  • an expression library such as a ⁇ gtl l cDNA expression library. can be screened with a labelled DNA primase polypeptide to identify cDNAs encoding polypeptides which specifically bind to the DNA primase polypeptide.
  • cDNA libraries usually comprise mammalian cDNA populations, typically human, mouse, simian, or rat. and may represent cDNA produced from RNA of one or more cell types, tissues, or organs at any of a variety of developmental. stage(s).
  • Specific binding for screening cDNA expression libraries is usually provided by including one or more blocking agent (e.g. , albumin, nonfat dry milk solids, etc.) prior to and/or concomitant with contacting the labeled DNA primase polypeptide (and/or labeled anti-DNA primase antibody).
  • blocking agent e.g. , albumin, nonfat dry milk solids, etc.
  • the invention also provides antisense polynucleotides complementary to polynucleotides encoding DNA primase polypeptide sequences and methods for identifying compounds comprising such nucleobase sequences that modulate DNA primase activity.
  • antisense polynucleotides are employed to inhibit transcription and/or translation of the DNA primase polypeptide mRNA species and thereby effect a reduction in the amount of the respective DNA primase polypeptide in a cell (e.g., a neoplastic cell of a patient).
  • DNA primase antisense polynucleotides are typically ssDNA, ssRNA, methylphosphonate backbone nucleic acids, phosphorothiolate backbone nucleic acids, polyamide nucleic acids, and the like antisense structures known in the art.
  • an antisense polynucleotide is administered to inhibit transcription and/or translation of DNA primase in a human immortal cell.
  • the present invention also provides a method for diagnosing a disease (e.g. , neoplasia. preneoplasia. senescence) in a human patient, wherein a diagnostic assay of human DNA primase activity as described herein is used to determine if a predetermined pathognomonic DNA primase activity level is present in a biological sample from a human or other patient; if the assay indicates the presence of DNA primase activity outside of the normal range or level (e.g.. within the predetermined pathognomonic activity range), the patient is diagnosed as having a disease condition or predisposition.
  • a disease e.g. , neoplasia. preneoplasia. senescence
  • the invention also provides therapeutic agents which inhibit neoplasia or cell replication by modulating function of DNA primase by inhibiting or augmenting formation of complexes of DNA primase:DNA polymerase or of DNA primase with other primase- binding polypeptides: such agents can be used as pharmaceuticals.
  • Such pharmaceuticals will be used to treat a variety of human and veterinary diseases, such as for example and not limitation: neoplasia, hyperplasia. benign prostatic hypertrophy, fibrocystic breast disease, reperfusion injury, myocardial infarction, stroke, traumatic brain injury, neurodegenerative diseases, aging, ischemia, toxemia, infection, autoimmune diseases. AIDS, hepatitis, and the like.
  • the invention provides methods of identifying DNA primase modulating agents by monitoring a heterodimerization assay.
  • a reaction mixture which includes (1) a DNA primase polypeptide capable of binding to a DNA polymerase species, (2) a DNA polymerase capable of binding to said DNA primase polypeptide under binding conditions, and (3) an agent is monitored for the ability of the agent to; inhibit heterodimerization of the DNA primase polypeptide species to the DNA polymerase.
  • Agents which inhibit said heterodimerization are DNA primase modulating agents. These DNA primase modulating agents can modulate apoptosis, cell proliferation, senescence, and/or cell differentiation. Such DNA primase modulating agents can serve as pharmaceuticals, commercial laboratory reagents, and solutes, among other uses.
  • the invention also provides diagnostic methods involving the use of a human DNA primase assay to diagnose and/or assist in the treatment of disease, such as cancer, and/or to identify pharmaceutical agents which can be used to treat cancer.
  • Figure 1 provides the chemical formulas for example primase modulators. DETAILED DESCRIPTION
  • an "activity" for a primase enzyme refers to a reaction mediated by the enzyme. This includes enzymatic synthesis of polynucleotides by the primase molecule (optionally in the presence of a polymerase). binding of the primase enzyme to a second molecule (e.g.. a polymerase molecule) and the like. This activity may be measured directly, e.g. , by monitoring incorporation of labeled nucleotides into a polynucleotide synthesized as a result of primase activity or formation of a primase-polymerase binding complex, or indirectly, e.g. , by monitoring binding of probes to polynucleotides synthesized as a result of primase activity, or primase dependent cell viability (e.g. , in a two hybrid system discussed below), or the like.
  • DNA primase refers to the mammalian DNA primase proteins designated generally in the art as p49 and p58, including isoforms thereof, unless otherwise stated: human and murine primase proteins and genes are preferred exemplifications of mammalian DNA primase.
  • an example DNA primase has substantial similarity to the reported sequences of the human and mouse p49 and p58 proteins. This substantial similarity is often at least 85 percent substantially identical to such reported sequences or are at least 90-95 percent substantially identical to the reported sequences, which can be found, for example in the following sources: for mouse primase: Prussak et al. (1989) J. Biol. Chem.
  • agent is used herein to denote a chemical compound (including, but not limited to, organic molecules, polynucleotides, proteins, peptides and the like), a mixture of chemical compounds, an array of spatially localized compounds (e.g., a peptide array, polynucleotide array, and/or combinatorial small molecule array; where "array” refers to a collection of different molecular species immobilized on a surface), a biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • a chemical compound including, but not limited to, organic molecules, polynucleotides, proteins, peptides and the like
  • array of spatially localized compounds e.g., a peptide array, polynu
  • Agents are evaluated for potential activity as antineoplastics, anti-infiammatories, or apoptosis modulators by inclusion in the described screening assays. Agents are evaluated for potential activity as primase modulators. Naturally occurring nucleotides, template polynucleotides, and ATP, all of which are reactants in the primase reaction, are not agents for the purposes of this invention.
  • protein interaction modulator is used herein to refer to an agent which can be identified by one or more screening method(s) of the invention as an agent which selectively modulates protein-protein binding between a first interacting polypeptide and a second interacting polypeptide. typically between primase and a second molecule.
  • Some protein interaction modulators have therapeutic potential as drugs for human use and/or serve as commercial reagents for laboratory research or bioprocess control.
  • anti-plastic agent is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human.
  • label refers to incorporation of a detectable marker, e.g.. by incorporation of a radiolabel in a nucleotide or amino acid or by attachment to a polypeptide or polynucleotide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods.
  • Various methods of labeling polypeptides and glycoproteins as well as nucleotides and polynucelotides are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to.
  • radioisotopes e.g., 3 H, 1 C. 35 S. 125 1. 131 I
  • fluorescent labels e.g., FITC. rhodamine. lanthanide phosphors
  • enzymatic labels e.g.. horseradish peroxidase. ⁇ - galactosidase, luciferase, alkaline phosphatase
  • biotinyl groups e.g., leucine zipper pair sequences, binding sites for secondary antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags.
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties.
  • substantially pure means an object species is a predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition, other than solvent, carrier, or other non- interfering substance), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all relevant macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (designated contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the terms “pathognomonic activity level” refers to a concentration, amount, or measured enzyme activity, respectively, of a mammalian DNA primase protein or primase/polymerase complex in a sample, that indicates the presence of a pathological (e.g. , neoplastic, senescent, immunodeficient, neurodegenerative, inflammatory. etc.) condition or a predisposition to developing a neoplastic disease, such as carcinoma, sarcoma, or leukemia.
  • pathological e.g. , neoplastic, senescent, immunodeficient, neurodegenerative, inflammatory. etc.
  • a predisposition to developing a neoplastic disease such as carcinoma, sarcoma, or leukemia.
  • such levels include any level necessary for the survival of a cell, such as a cancer cell, deleterious to the host organism.
  • a pathognomonic activity is a level in a cell or cellular sample that falls outside the range of normal clinical values that is established by prospective and/or retrospective statistical clinical studies.
  • neoplastic disease e.g., carcinoma, sarcoma, or leukemia
  • neoplastic cells can often exhibit an amount of DNA primase protein or mRNA in a cell or tissue sample that is outside the range of concentrations that characterize normal.
  • undiseased individuals typically the pathognomonic activity is at least about one standard deviation outside the mean normal value, more usually it is at least about two standard deviations or more above the mean normal value.
  • Clinical diagnostic tests can produce some percentage of false positives and false negatives.
  • diagnostic assays of the invention can be adjusted to satisfy the diagnostic objective and any relevant regulatory requirements of the particular application.
  • diagnostic methods of the invention are used to identify individuals as having a disease, and provide an additional parameter in a differential diagnosis of disease made by a competent health professional.
  • certain cancer cells can have substantially the same, substantially more, or substantially less DNA primase activity than a similar non-cancerous cell; nonetheless, DNA primase inhibitors would be suitable to treat such cancers.
  • physiological conditions refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.
  • the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.
  • Suitable in vitro reaction conditions for in vitro transcription reaction mixtures are generally physiological conditions.
  • in vitro physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5. 20-45°C and 0.001-10 mM divalent cation (e.g., Mg + + , Ca + + ); preferably about 150 mM NaCl or KC1, pH 7.2-7.6.
  • aqueous conditions 5 M divalent cation, and often include 0.01-1.0 percent nonspecific protein (e.g.. BSA).
  • a non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to 2% , typically 0.05-0.2% (v/v).
  • Particular aqueous conditions may be selected by the practitioner according to conventional methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl. 5-50 mM Tris HCI. pH 5-8. with optional addition of divalent cation(s) and/or: metal chelators: nonionic detergents: membrane fractions: antifoam agents; and/or scintillants.
  • interacting polypeptide segment and "interacting polypeptide sequence” refer to a portion of a protein (naturally-occurring or hybrid) which can form a specific binding interaction with a portion of a second protein under suitable binding conditions.
  • a portion of the first protein preferentially binds to a portion of the second protein forming a heterodimer or higher order heteromultimer comprising the first and second hybrid proteins; the binding portions of each hybrid protein are termed interacting polypeptide segments.
  • interacting polypeptides can form heterodimers with a dissociation constant (K D ) of about 1 x 10 1 M 1 , usually about 1 x 10 4 M 1 , typically about 1 x 10 s M 1 , preferably at least 1 x lf M-' to 1 x 10 7 M-' or less, under suitable physiological conditions.
  • K D dissociation constant
  • recombinant refers to a polypeptide (e.g. , DNA primase and/or DNA polymerase) produced by recombinant DNA techniques wherein the gene coding for protein is cloned by recombinant DNA technology.
  • the human gene for DNA primase may be inserted into a suitable DNA vector, such as a bacterial vector or eukaryotic host cell expression vector, and the vector used to transform a suitable host. The gene is then expressed in the host to produce the recombinant protein or other product.
  • the transformed host may be prokaryotic or eukaryotic, including mammalian, yeast, Aspergillus and insect cells.
  • One preferred embodiment employs bacterial cells as the host.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been otherwise intentionally modified by man in the laboratory is naturally-occurring.
  • naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.
  • neoplasia is characterized by a clonally derived cell population which has a diminished capacity for responding to normal cell proliferation control signals.
  • Oncogenic transformation of cells leads to a number of changes in cellular metabolism, physiology, and morphology.
  • One characteristic alteration of oncogenically transformed cells is a loss of responsiveness to constraints on cell proliferation and differentiation normally imposed by the appropriate expression of cell growth regulatory genes, leading to undesired cell proliferation.
  • the precise molecular pathways and secondary changes leading to malignant transformation for many cell types is not completely clear. However, for cells to replicate and form viable progeny it is essential that DNA replication occur.
  • agents which can modify the activity(ies) of the replicative proteins involved in DN. ⁇ replication so as to be able to modulate cell proliferation, differentiation, and/or apoptosis for therapeutic or prophylactic benefit.
  • agents can serve as commercial research reagents for control of cell proliferation, differentiation, and/or apoptosis in experimental applications, and/or for controlled proliferation and differentiation of predetermined cells (e.g. , hematopoietic stem cell populations) in vitro, in ex vivo therapy, or in vivo.
  • predetermined cells e.g. , hematopoietic stem cell populations
  • DNA primase modulating agents can provide novel chemotherapeutic agents for treatment of neoplasia, lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases, and the like. Moreover, the ability to screen for such compounds is of immediate commercial benefit to pharmaceutical and drug discovery companies.
  • DNA Primase and Polymerase Polypeptides and Polynucleotides The nomenclature used herein, reagents and laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization utilized in the practice of the invention involve certain procedures known and commonly employed in the art.
  • the known full coding sequences for murine and human DNA primase subunits and DNA polymerase proteins make possible the construction and isolation of polynucleotides that can direct the expression of recombinant DNA primase and polymerase. fragments thereof, or analogs thereof.
  • primase activity is found in nuclear extracts from cells, e.g. , prepared by isolation of the cellular nuclei and lysis of the nuclei. Nuclei are isolated by known techniques, including cell lysis, centrifugation and the like.
  • Polynucleotides encoding full-length DNA primase or fragments or analogs thereof may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences, such that the encoded polypeptide product is produced.
  • RNA, DNA, cDNA, genomic DNA, genomic RNA or a hybrid of the various combinations are isolated from natural sources, cloned heterologous sources, or synthesized in vitro.
  • the nucleic acids are present in transduced or transfected whole cells, in transduced or transfected cell lysates.
  • RNA endonucleases i.e. , where the RNA is a ribozyme
  • RNA endonucleases i.e. , where the RNA is a ribozyme
  • RNA endonucleases i.e. , where the RNA is a ribozyme
  • RNA polymerase mediated techniques e.g. , NASBA
  • Oligonucleotides useful as probes in the assays of the invention are synthesized on an automated synthesizer such as an Applied Bio Systems oligonucleotide synthesizer, according to specifications provided by the manufacturer. Production of anti-DNA Primase or anti-Pol Antibodies
  • Native primase and polymerase proteins, fragments thereof, or analogs thereof, may be used to immunize an animal for the production of specific antibodies.
  • antibodies can comprise a polyclonal antiserum or can comprise a monoclonal antibody produced by hybridoma cells.
  • Antibodies A Laboratory Manual. (1988) E. Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. These antibodies are optionally used to monitor the presence or absence of primase in an assay of the invention.
  • the invention provides hybrid screening assays and related host organisms (typically unicellular organisms) which harbor a mammalian DNA primase protein two- hybrid system, typically in the form of polynucleotides encoding a first hybrid protein, a second hybrid protein, and a reporter gene, wherein said polynucleotide(s) are either stably replicated or introduced for transient expression.
  • the host organism is a yeast cell (e.g. , Saccharomvces cervisiae) in which the reporter gene transcriptional regulatory sequence comprises a Gal4-responsive promoter.
  • Yeast comprising (1) an expression cassette encoding a GAL4 DNA binding domain (or GAL4 activator domain) fused to a binding fragment of primase capable of binding to a DNA polymerase polypeptide, (2) an expression cassette encoding a GAL4 DNA activator domain (or GAL4 binding domain, respectively) fused to a member of a cDNA library or a binding fragment of a DNA polymerase capable of binding to a mammalian DNA primase polypeptide, and (3) a reporter gene (e.g., 0-galactosidase) comprising a cis-linked GAL4 transcriptional response element can be used for agent screening.
  • a reporter gene e.g., 0-galactosidase
  • Such yeast are incubated with a test agent and expression of the reporter gene (e.g., ⁇ -galactosidase) is determined; the capacity of the agent to inhibit expression of the reporter gene as compared to a control culture identifies whether the candidate agent is a DNA primase modulatory agent.
  • the reporter gene e.g., ⁇ -galactosidase
  • Yeast two-hybrid systems may be used to screen a mammalian (typically human) cDNA expression library, wherein human cDNA is fused to a GAL4 DNA binding domain or activator domain, and either a DNA primase or DNA polymerase polypeptide sequence is fused to a GAL4 activator domain or DNA binding domain, respectively.
  • a yeast two-hybrid system can screen for cDNAs that encode proteins which bind to DNA primase or polymerase sequences.
  • a cDNA library can be produced from mRNA from a human mature B cell (Namalwa) line (Ambrus et al. (1993) Proc. Natl. Acad. Sci.
  • Such a cDNA library cloned in a yeast two-hybrid expression system can be used to identify cDNAs which encode proteins that interact with primase or polymerase and thereby produce expression of the GAL4-dependent reporter gene.
  • Polypeptides which interact with primase or polymerase can also be identified by immunoprecipitation of primase or polymerase with antibody and identification of co- precipitating species and by screening a peptide library (e.g., a bacteriophage peptide display library, a spatially defined peptide array, and the like) with a primase or polymerase polypeptide.
  • a peptide library e.g., a bacteriophage peptide display library, a spatially defined peptide array, and the like
  • yeast two-hybrid systems The construction of yeast two-hybrid systems is generally known. This approach identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator (Fields and Song (1989) Nature 340: 245). such as the yeast Gal4 transcription protein.
  • the yeast Gal4 protein which consists of separable domains responsible for DNA-binding and transcriptional activation. Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell.
  • Intermolecuiar binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) which is operably linked to a Gal4 binding site.
  • a reporter gene e.g., lacZ, HIS3
  • the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver SC and Hunt SW (1993) Mol. Biol.
  • Administration of an efficacious dose of an agent capable of specifically inhibiting primase:polymerase complex formation to a patient can be used as a therapeutic or prophylactic method for treating pathological conditions (e.g., cancer, inflammation, lymphoproiiferative diseases, autoimmune disease, neurodegenerative diseases, and the like) which are effectively treated by modulating DNA replication.
  • pathological conditions e.g., cancer, inflammation, lymphoproiiferative diseases, autoimmune disease, neurodegenerative diseases, and the like
  • assays which monitor primase-polymerase binding are of value in screening for primase modulators.
  • binding assays often take one of two forms: immobilized primase polypeptide(s) can be used to bind labeled polymerase polypeptide(s), or conversely, immobilized polymerase polypeptide(s) can be used to bind labeled primase polypeptides.
  • the labeled polypeptide is contacted with the immobilized polypeptide under conditions that permit specific binding of the polypeptides(s) to form a complex in the absence of added agent.
  • Particular aqueous conditions may be selected by the practitioner according to conventional methods.
  • buffered aqueous conditions may be used: 10-250 mM NaCl, 5-50 mM Tris HCI, pH 5-8, with optional addition of divalent cation(s) and/or metal cheiators and/or nonionic detergents and/or membrane fractions. Additions, deletions, modifications (such as pH) and substitutions (such as KC1 substituting for NaCl or buffer substitution) may be made to these basic conditions. Modifications can be made to the basic binding reaction conditions so long as specific binding of primase polypeptide(s) to polymerase polypeptides occurs in the control reaction(s).
  • At least one polypeptide species typically is labeled wiui a detectable marker.
  • Suitable labeling includes, but is not limited to. radiolabeiing by incorporation of a radiolabeled amino acid (e.g. , C-labeled leucine, 3 H-labeled glycine, 35 S- labeled methionine), radiolabeling by post-translational radioiodination with I25 I or 131 I (e.g., Bolton-Hunter reaction and chloramine T), labeling by post-translational phosphorylation with 32 P (e.g., phosphorylase and inorganic radiolabeled phosphate) fluorescent labeling by incorporation of a fluorescent label (e.g., fluorescein or rhodamine), or labeling by other conventional methods known in the art.
  • a fluorescent label e.g., fluorescein or rhodamine
  • the other polypeptide is generally labeled with a detectable marker.
  • a primase or polymerase polypeptide may be used in combination with an accessory protein (e.g., a protein which forms a complex with the polypeptide in vivo). It is typically preferred that different labels are used for each polypeptide species, so that binding of individual and/or heterodimeric and/or multimeric complexes can be readily distinguished.
  • an accessory polypeptide e.g., a protein which forms a complex with the polypeptide in vivo.
  • a primase polypeptide is labeled with fluorescein and an accessory polypeptide is labeled with a fluorescent marker that fluoresces with either a different excitation wavelength or emission wavelength, or both.
  • double-label scintillation counting is used, wherein a primase polypeptide is labeled with one isotope (e.g., 3 H) and a second polypeptide species is labeled with a different isotope (e.g. , 14 C) that can be distinguished by scintillation counting using standard discrimination techniques.
  • Labeled polypeptide(s) are contacted with immobilized polypeptide(s) under aqueous conditions as described herein.
  • the time and temperature of incubation of a binding reaction is optionally varied, with the selected conditions permitting specific binding to occur in a control reaction where no agent is present.
  • Preferable embodiments employ a reaction temperature of about at least 15 degrees Centigrade, more preferably 30 to 42 degrees Centigrade, and a time of incubation of approximately at least 15 seconds, although longer incubation periods, from 30 seconds to a minute to several minutes or more, are preferable so that, in some embodiments, a binding equilibrium is attained.
  • Binding kinetics and the thermodynamic stability of bound primase: polymerase complexes determine the latitude available for varying the time, temperature, salt, pH, and other reaction conditions.
  • desired binding reaction conditions can be calibrated readily by the practitioner using conventional methods in the art, which may include binding analysis using Scatchard analysis. Hill analysis, and other standard analytic methods (Proteins. Structures and Molecular Principles.
  • the aqueous phase containing non-immobilized protein is removed and the substrate containing the immobilized polypeptide species and any labeled protein bound to it is washed with a suitable buffer, optionally containing unlabeled blocking agent(s), and the wash buffer(s) removed.
  • a suitable buffer optionally containing unlabeled blocking agent(s)
  • the wash buffer(s) removed.
  • the amount of detectable label remaining specifically bound to the immobilized polypeptide is determined (e.g. , by optical, enzymatic, autoradiographic, or other radiochemical methods).
  • unlabeled blocking agents that inhibit non-specific binding
  • blocking agents include, but are not limited to. the following: calf thymus DNA, salmon sperm DNA, yeast RNA, mixed sequence (random or pseudorandom sequence) oligonucleotides of various lengths, bovine serum albumin, nonionic detergents (NP-40, Tween. Triton X-100, etc.), nonfat dry milk proteins, Denhardt's reagent, polyvinylpyrrolidone, Ficoll. and other blocking agents.
  • blocking agents at suitable concentrations to be included in binding assays; however, reaction conditions are selected so as to permit specific binding between a primase polypeptide and a polymerase polypeptide in a control binding reaction.
  • Blocking agents are included to inhibit nonspecific binding of labeled protein to immobilized protein and/or to inhibit nonspecific binding of labeled polypeptide to the immobilization substrate.
  • covalent or noncovalent linkage to a substrate may be used.
  • Covalent linkage chemistries include, but are not limited to, well-characterized methods known in the art (Kadonaga and Tijan (1986) Proc. Natl. Acad. Sci. (U.S.A.) 83: 5889).
  • One example, not for limitation, is covalent linkage to a substrate derivatized with cyanogen bromide (such as CNBr-derivatized Sepharose 4B).
  • Noncovalent bonding of proteins to a substrate include, but are not limited to, bonding of the protein to a charged surface (e.g. , on a bead) and binding with specific antibodies.
  • parallel binding reactions. are conducted. wherein one set of reactions serves as control and at least one other set of reactions include various quantities of agents, mixtures of agents, or biological extracts, that are being tested for the capacity to inhibit binding of a primase polypeptide to a polymerase polypeptide or disrupt, modulate, inhibit, or potentiate the activity of either or both.
  • primase inhibitors agents which, when added to a binding reaction, inhibit formation of primase:polymerase complexes are thereby identified as primase inhibitors; such agents inhibit DNA replication and can be used to inhibit replication of neoplastic cells.
  • Agents which, when added to a binding reaction, enhance formation of primase:polymerase complexes are thereby identified as primase potentiators (e.g., primase agonists; such agents can find use to enhance DNA replicative potential and cell viability, including viability of senescent cell types.
  • primase potentiators e.g., primase agonists; such agents can find use to enhance DNA replicative potential and cell viability, including viability of senescent cell types.
  • several binding reactions are monitored simultaneously, e.g. , using a format which permits simultaneous analysis of several samples (microtiter plates, etc.).
  • the assays are automated, e.g. , using robotics for pipetting samples into microtiter plates.
  • One means for detecting binding of a primase polypeptide to a polymerase polypeptide is to immobilize the primase polypeptide. such as by covalent or noncovalent chemical linkage to a solid support, and to contact the immobilized primase polypeptide with a polymerase polypeptide that has been labeled with a detectable marker (e.g., by incorporation of radiolabeled amino acid, by epitope tagging and reporting with a fluorescent-labelled anti-epitope tag antibody, and the like).
  • a detectable marker e.g., by incorporation of radiolabeled amino acid, by epitope tagging and reporting with a fluorescent-labelled anti-epitope tag antibody, and the like.
  • Such contacting is typically performed in aqueous conditions which permit binding of a primase polypeptide to a polymerase polypeptide. Binding of the labeled polymerase polypeptide to the immobilized primase is measured by determining the extent to which the labeled polymerase polypeptide is immobilized as a result of a specific binding interaction. Such specific binding may be reversible, or may be optionally irreversible if a cross-linking agent is added in appropriate experimental conditions.
  • the binding assay is performed in vivo in a cell, such as a yeast cell (e.g., Saccharomvces). and agents which inhibit intermolecular binding between a primase protein and a polymerase polypeptide are identified as primase-modulating agents.
  • a cell such as a yeast cell (e.g., Saccharomvces).
  • agents which inhibit intermolecular binding between a primase protein and a polymerase polypeptide are identified as primase-modulating agents.
  • the in vivo screening assay is optionally a yeast two-hybrid system wherein the yeast cells express: (1) a first fusion protein comprising primase and a first transcriptional regulatory protein sequence (e.g.
  • GAL4 activation domain (2) a second fusion protein comprising a polymerase polypeptide and a second transcriptional regulatory protein sequence (e.g., GAL4 DNA-binding domain), and (3) a reporter gene (e.g., ⁇ - galactosidase, an auxotroph complementing gene) which is transcribed when an intermolecular complex comprising the first fusion protein and the second fusion protein is formed.
  • a functional primase:polymerase polypeptide complex forms, such as in a control assay lacking agent, the cell expresses the reporter gene which can be detected.
  • Agents which inhibit or augment formation of functional primase:polymerase polypeptide complexes are thereby identified as primase-modulating agents and candidate drugs and commercial cell culture reagents and cell preservatives and the like.
  • the physical interaction of the bound labeled complex with the surface is reported, such as where the surface is a fluor or scintillant and the label in the bound labeled complex emits radiation suitable for activating the fluor or scintillant of the surface; light emitted from the surface reports the relative amount of bound labeled complex.
  • a suitable system is the scintillation proximity assay (Amersham), wherein the unlabeled component is bound to a fluor-containing bead.
  • Alternative systems include the "Flash Plate” system (LKB).
  • the DNA primase is labeled with a first fluor which absorbs radiation (particle or wave) and emits phosphorescent or fluorescent light at a first wavelength
  • the DNA polymerase is labeled with a second fluor which absorbs radiation at said first wavelength and thereby emits fluorescent or phosphorescent radiation at a second wavelength.
  • the labeled DNA primase and DNA polymerase are incubated under suitable binding conditions, and at suitable reactant concentrations whereby the amount of radiation of the second wavelength is approximately proportional to the amount of bound primase/polymerase complexes, and excited with radiation of the first wavelength (or particle type) and the amount of emitted radiation of the second wavelength is detected.
  • the relative amount of radiation of the second wavelength reports the relative amount of bound DNA primase/DNA polymerase complexes.
  • suitable system is a dye-dye transfer system (Packard). Agents are evaluated to determine their capacity to modulate (i.e.. inhibit or potentiate) the intermolecular binding of primase and polymerase in comparison to a control reaction lacking the agent. Active agents can then be tested for their capacity to inhibit DNA primase/DNA polymerase activity in a coupled activity assay as described herein.
  • the DNA primase and primase/polymerase assays employed in the present method can be performed by any suitable method known in the art for measuring mammalian DNA primase and/or coupled primase/polymerase activity.
  • Preferred primase template polynucleotides and reaction conditions include, for example, those described in Suzuki et al. (1993) Biochemistry 32: 12782; Kuchta et al. (1992) Biochemistry 31: 4720; Copeland and Wang (1993) J. Biol. Chem. 268: 26179; Harrington and Perrino (1995) Nucl. Acids Res. 23; 1003, each incorporated herein by reference.
  • the Examples herein provide additional preferred embodiments.
  • primase reactions optionally comprise the following reaction conditions: 100 pmol of a 40mer DNA template comprising all 4 nucleotides; 20 mM Tris, pH 7.5; 5 mM MgC12; 2 mM DTT; 0.1 mg/ml BSA; 100 ⁇ M each of ATP, CTP, GTP, and UTP, wherein one of the NTPs is labeled at the phosphate with -P; and pol ⁇ :primase (0.7U pol and 6.3U primase). Incubation is typically at 25-
  • the amount of a product polynucleotide is determined by assessing the reaction mixture, e.g. , following a suitable incubation period with a substrate which selectively immobilizes or binds to polynucleotides and which substantially does not immobilize or bind to mononucieotides; one example of such a substrate is a charged membrane (e.g.. Nylon 66, nitrocellulose, DEAE paper, whatman paper, filter paper, ere).
  • a charged membrane e.g.. Nylon 66, nitrocellulose, DEAE paper, whatman paper, filter paper, ere.
  • reaction products can be chromatographed or electrophoresed (e.g.. PAGE) to separate polynucleotide products from unincorporated nucleotides.
  • PAGE electrophoresed
  • coupled primase/polymerase reactions can comprise the following reaction conditions: 100 pmol of a linear ssDNA template comprising all 4 nucleotides and being about 450 nt in length; 20 M Tris, pH 7.5; 5 mM MgC12; 2 mM ATP; 200 ⁇ M CTP, GTP, and UTP; 100 ⁇ M dATP, dGTP, dTTP, and 25 ⁇ M 32 P- ⁇ -dCTP; and pol ⁇ rimase (9.3U pol a and 29 primase). Incubation is typically at 25-42 degrees C. with a preferred temperature being about 30-37 degrees C. Incubation times are often .5 minutes to 60 minutes.
  • reactions are conveniently stopped by addition of EDTA to 5 mM final concentration.
  • the amount of product polynucleotide is determined by contacting the reaction mixture, following a suitable incubation period, with a substrate which selectively immobilizes or binds to polynucleotides and which substantially does not immobilize or bind to mononucleotides; one example of such a substrate is a charged membrane (e.g. , Nylon 66, nitrocellulose, DEAE paper whatman paper, filter paper, ere).
  • reaction products can be chromatographed or electrophoresed (e.g., PAGE) to separate polynucleotide products from unincorporated nucleotides.
  • the amount of label incorporated into polynucleotide products report the relative activity of the DNA primase.
  • Compound Chemistry The invention provides several example primase modulators.
  • the primase modulator compounds shown in Figure 1 are easily synthesized by one of skill using widely available compounds, given the structure of the particular modulator, using standard synthetic techniques. A guide to standard synthetic organic chemistry is found in March. Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed J. Wiley and Sons (New York, 1992) and the references cited therein.
  • additional primase modulators are easily synthesized by reference to the given primase modulators by minor modification of the example modulators.
  • alkyl groups branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical having from 1-12 carbons
  • Substituted alkyls an alkyl having one or more functional group such as a lower alkyl. aryl, acyl. halogen (i.e., alkylhalos, e.g.. CF-,), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy. aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like) are also substituted for one another using standard synthetic methods.
  • Aryls (aromatic substituents such as single or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety) can be substituted at similar positions in the given primase modulators.
  • Typical aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
  • substituted aryls including an aryl and including one or more functional group such as a lower alkyl, acyl. halogen, alkylhalos, hydroxy, amino, alkoxy.
  • alkylamino, acylamino, acyloxy, mercapto and both saturated and unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety are optionally substituted for one another using available techniques.
  • aryl or substituted aryl are also substituted for one another.
  • Halogens, including fluorine, bromine, chlorine and iodine atoms are substituted at similar positions on the given modulators to yield functionally similar molecules.
  • Hydroxy groups are optionally substituted with primary amines (R— NH 2 ) or Alkoxy groups (an —OR group, where R is a lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or substituted arylaikyl.
  • Alkylamino groups are substituted, in which secondary and tertiary amines wherein the alkyl groups may be either the same or different and may consist of straight or branched, saturated or unsaturated hydrocarbons are substituted for one another.
  • Mercapto groups having the general structure R— S— R' wherein R and R' are the same or different and are alkyl, aryl or heterocyclic as described herein are optionally subsituted for one another.
  • Saturated cyclic hydrocarbons such as cyclopropyl, cyclobutyl, cyclopentyl, etc. , and substituted analogues of these structures are optionally substituted for one another.
  • Unsaturated cyclic hydrocarbons (a monovalent non-aromatic group with at least one double bond, such as cyclopentene. cyclohexene. etc. and substituted analogues thereof are optionally substituted for one another in the modulators of the invention.
  • Heteroaryl groups having aromatic rings in which one or more carbon atoms of the aromatic ring(s) are substituted by a heteroatom such as nitrogen, oxygen or sulfur with a single aromatic ring, multiple aromatic ring(s), or one or more aromatic rings coupled to one or more non-aromatic ring(s) can be substituted for one another using standard organic chemical synthetic methods.
  • the rings can be fused together, linked covalently. or linked to a common group such as a methylene or ethylene moiety.
  • Substituted heteroaryl groups having one or more functional group such as lower alkyl. acyl, halogen, alkylhalos (e.g. CF 3 ), hydroxy, amino.
  • heterocyclic groups having a monovalent saturated or unsaturated non-aromatic group having a single ring or multiple condensed rings from 1-12 carbon atoms and from 1-
  • the present invention utilizes a nuclear extract to obtain primase enzyme, using a CHAPS nuclear extract protocol.
  • Hek 293 cells were used as a source of cells for preparation of nuclei.
  • HEK 293 cells are an adenovirus-transformed human embryonic kidney cell line. These cells grow readily in suspension cultures with an optimal harvest density of 0.5 X 10 9 cells per liter and doubling time of 24 hours.
  • a commercial supplier, Cellex maintains 293 suspension cultures in spinner flasks, and provides a weekly supply of 2 X 10" cells (from 400 liters of culture).
  • wash buffer (lOmM HEPES, pH 7.5. 1.5 mM MgCl 3 , 10 mM KCL, ImM DTT) was added to 1 volume of packed cells.
  • the cells were partially mixed in the wash buffer using gentle mixing to avoid lysis of the cells.
  • the cells were pelleted by centrifugation at 1780 x g for 10 minutes at 4 C C. The supernatant was discarded and 5 volumes of lysis buffer (10 M Tris HCL, pH 7.5. 1.5 mM MgCl 3 , 10 mM KCL, ImM DTT) was added to 1 volume of packed cells.
  • the cells were partially mixed in the wash buffer using gentle mixing to avoid lysis of the cells.
  • the cells were pelleted by centrifugation at 1780 x g for 10 minutes at 4 C C.
  • the supernatant was discarded and 5 volumes of lysis buffer (10 M Tris HCL, pH
  • nuclear extract buffer (20 M HEPES pH 7.9, 20 mM NaCl, 1.5 mM MgCl 2 , .5 M EGTA, 25% glycerol. and. added just before use, 1 mM DTT, .1 mM PMSF, ImM sodium metabisulflte and ImM Benzamidine). While vortexing, 0.5 volumes of nuclear extract buffer containing 1.2 M NaCl was added. The mixture was dounce homogenized, placed on ice and stirred for at least 30 minutes. The mixture was then spun for 75 minutes at 18,000 rpms in an SS34 rotor.
  • primase assay measuring primase activity by incorporation of radioactive nucleotides is described.
  • primase was diluted with primase dilution buffer (50% glycerol.
  • a typical 60 ⁇ l reaction volume further included 50 mM Tris HCI (pH 8.0); 20 mM KCl, 200 ⁇ g/ml BSA, 4mM MgCl 2 . 2 mM fresh DTT, 2 mM rATP, 5 uCi 32 P (800 Ci/mmol) or 2.5 ⁇ Ci 33 P (2,000 Ci/mMol) dATP, 10 ug/mi Poly (dT), and primase and polymerase a.
  • the recombinant 180 Kd human poiymerase protein was isolated from a bacculovirally transduced SF9 cell as described in Wang et al (1995) Methods in Enzvmology 262: 77-84. Typically, about .05 ⁇ g of pl80 and about .025 ⁇ g of a 1 : 1 mixture of p49/p58 primase subunits were used per reaction. Several reactions were typically set up in parallel, although a single reaction can also be assessed. For example, a Zymate XP (Zymark Corporation; Hopkinton.
  • Example 3 A Primase Assay Using Nuclear Extract as A Source for Primase Activity. An assay similar to Example 2 was also developed to measure primase activity.
  • the assay is described below with reference to using a nuclear extract as the source for primase activity, one of skill will recognize that the assay can easily be adapted for purified or recombinant primase. Examples 1 and 2 provide a description of the buffers, nuclear extracts, etc.
  • Example 4 Primase Modulators
  • the primase modulator compounds show in Figure 1 were determined to modulate primase activity in the assays shown above, using a nuclear extract comprising primase activity, as described above.
  • the example modulators are primase inhibitors.
  • the IC50s for the compounds indicated in Figure 1 was the approximate concentration of the modulator at which 50% of the primase enzyme activity in a reaction mixture was inhibited by the modulator.

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Abstract

L'invention concerne des méthodes de criblage de l'ADN primase permettant d'identifier des agents modulateurs de l'ADN primase, des méthodes permettant de moduler l'activité de l'ADN primase et des compositions modulant l'ADN primase.
PCT/US1997/004654 1997-03-21 1997-03-21 Cribles d'adn primase de mammifere et agents modulateurs d'activite WO1998042869A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000041524A2 (fr) * 1999-01-11 2000-07-20 President And Fellows Of Harvard College Amplification isotherme d'adn

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5360714A (en) * 1992-08-28 1994-11-01 Fox Chase Cancer Center Hepadnavirus polymerase gene product having RNA-dependent DNA priming and reverse transcriptase activities and methods of measuring the activities thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5360714A (en) * 1992-08-28 1994-11-01 Fox Chase Cancer Center Hepadnavirus polymerase gene product having RNA-dependent DNA priming and reverse transcriptase activities and methods of measuring the activities thereof

Non-Patent Citations (2)

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Title
CATAPANO C.V. et al., "Inhibition of Primer RNA Formation in CCRF-CEM Leukemia Cells by Fludarabine Triphosphate", CANCER RES., 01 April 1991, Vol. 51, No. 7, pages 1829-1835. *
SHEAFF R.J., "Calf Thymus DNA Polymerase alpha-Primase: 'Communication' and Primer-Template Movement Between the Two Active Sites", BIOCHEMISTRY, 1994, Vol. 33, No. 8, pages 2247-2254. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000041524A2 (fr) * 1999-01-11 2000-07-20 President And Fellows Of Harvard College Amplification isotherme d'adn
WO2000041524A3 (fr) * 1999-01-11 2001-02-08 Harvard College Amplification isotherme d'adn

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