WO1999049043A2 - Human rad17 cell cycle checkpoint - Google Patents

Abstract

Disclosed are novel checkpoint polynucleotides encoding polypeptides designated rad17, polypeptides encoded by the polynucleotides, expression constructs comprising the polynucleotides, host cells transformed or transfected with the polynucleotides, methods for producing the polypeptides, antibodies immunospecific for the polypeptides, methods to identify binding partners of the polypeptides, and methods to screen for modulators of rad17 biological activity.

Description

Human Radl7 Cell Cycle Checkpoint BACKGROUND

The process of eukaryotic cell growth and division is the somatic, or mitotic, cell cycle which consists of four phases, the Gl phase, the S phase, the G2 phase, and the M phase. During the Gl (gap) phase, biosynthetic activities of the cell are at a high rate. The S (synthesis) phase begins when DNA synthesis starts and ends when the DNA content of the nucleus of the cell has been replicated and two identical sets of chromosomes are formed. The cell then enters the G2 (gap) phase which continues until mitosis starts. In mitosis, the chromosomes pair and separate and two new nuclei form, and in cytokinesis, the cell splits into two daughter cells, each receiving one nucleus containing one of the two sets of chromosomes. Mitosis and cytokinesis together make up the M (mitosis) phase of the cell cycle. Cytokinesis terminates the M phase. The sequence in which the events of the cell cycle proceed are tightly regulated such that the initiation of one cell cycle event is dependent on the completion of the prior cell cycle event. This control allows fidelity in the duplication and segregation of genetic material from one generation to the next.

The term "cell cycle checkpoint" refers to the proteins, signals, processes, and feedback control that integrate discontinuous events during cellular replication in order to maintain essential dependencies within the cell cycle. See generally, Elledge, Science 274: 1664-1612 (1996) and Nurse, Cell 91:.865-867

(1997). Failure of the cell cycle checkpoints predisposes individuals to, or directly causes, many disease states such as cancer, axatia telangiectasaia, embryo abnormalities, and various immunological defects associated with aberrant B and T cell development. The latter are associated with pathological states such as lupus, arthritis, and autoimmune diseases. Intense research efforts have therefore focused on identifying cell cycle checkpoints and the proteins essential for the function of the checkpoints.

Genetic analysis in the yeast S. pombe and S. cerevisiae has identified a number of genes important for cell cycle arrest and DNA repair essential to regulating cell cycle progression. For a review, see Carr and Hoekstra, Trends in Cell Biology

5:32-40 (1995). One such gene identified in both yeast species is required for a DNA damage checkpoint which arrests the cell cycle at the G2 phase as well as a related checkpoint which monitors the completion of DNA synthesis and arrests the cell cycle at the S phase The gene identified in S pombe has been designated radl 7-¹- and in S cerevisiae, the corresponding gene has been designated RAD17 [Paulovich. et al, Genetics 145 45-62 (1997), Siede et al, Nucl. Acids Res. 24.1669-1615 (1996)]

Cells having mutations in radl 7 fail to either sense or appropriately respond to DNA damage and subsequently lose viability more rapidly than wild type cells after exposure to clastogenic agents or events (e g , radiation, DNA damaging agents, and the like) Sensitivity to radiation can cause defects in checkpoint responses or defects in direct DNA repair reactions

The product of the S pombe radl 7+ gene is an approximately 69 kDa proteins that shares sequence similarity to subunits of the mammalian protein RF-C RF-C is a five subunit processivity factor involved in DNA replication and all five subunits share sequence similarity to each other All five subunits, as well as radl 7+ and RAD24, have the canonical Walker box sequences that are indicative of ATPase activity RF-C acts to recognize primer-template junctions at origins of DNA replication At these sites, RF-C binds and subsequently facilitates the loading of proliferating cell nuclear antigen (PCNA) and DNA polymerase, suggesting that RAD 17 acts to sense DNA damage during cell cycle checkpoint responses Kuerbitz, et al., Proc. Natl. Acad. Sci.fUSA) 89 74932-7495 (1992) disclosed that the tumor suppressor protein p53 is required for a Gl checkpoint and cell cycle arrest observed following DNA damage Radiation of cells was shown to result in increased levels of p53 leading to the transcriptional activation of p53 responsive genes One such p53-induced target is the product of the WAFl gene (also called p21, CIPl, and sidl) WAFl is a member of an expanding class of cell cycle regulatory proteins termed cyclin-dependent kinase inhibitory proteins which control transition through the cell cycle Transcriptional activation of WAFl thus provides a direct link between DNA damage-dependent induction of p53 and the inhibition of kinases essential for cell cycle progression See Elledge and Harper, Current Opinions in Cell Biology 6:847-852 (1994) Mutations in the p53 gene are one of the most common genetic alteration in human cancers For example, Baker, et al, Cell 61.159- 767 (1990) reported that breast, lung, bladder, and brain tumors have been associated with the loss of chromosome 17p, the region to which the p53 gene has been localized.

At present, there is relatively little known about the molecular components of the G2 checkpoint in mammals. Caffeine is a chemical entity that abrogates G2 checkpoint control [Russell, et al, Cancer Res. 55: 1639-1642 (1995)]. Powell, et al. Cancer Res 55: 1643-1649 (1995) also reported that analysis of cell lines which differ only by the presence or absence of a functional p53 demonstrated preferential caffeine-enhanced sensitization to radiation in those cells lacking the p53- dependent checkpoint. Thus, potentially lethal damage is greater in cells lacking the Gl and G2 checkpoints in comparison to cells containing an intact Gl checkpoint. While certain cells undergo DNA damage-dependent cell cycle arrest, other cells appear to respond to DNA damage by initiating an intrinsic suicide program termed apoptosis or programmed cell death. The factors determining which process occurs are not full understood. Recent work has demonstrated an important role for p53 both in the regulation of Gl cell cycle transitions and apoptosis. Symonds, et al, cell 78:103-111 (1994) described p53-dependent apoptosis as suppressing tumor growth and progression in vivo.

High doses of radiation and chemotherapy are used to treat tumor cells in order to damage DNA so severely that the cells will die. However, even though tumor cells having a mutation in the p53 gene are defective in a Gl checkpoint, they can still repair DNA damage induced by irradiation or chemotherapy.

Thus there exists a need in the art for identification of the mammalian proteins that are involved in the cell cycle checkpoints in order to develop therapies for the human disease states associated with defective cell cycle checkpoints and for the use of the isolated genes encoding those proteins which in themselves may be useful as therapies. Identification of these proteins, and their underlying genes, would enable the development of therapeutically useful modulators of the proteins encoded by the genes. Inhibition of a radl -dependent checkpoint in tumors cells could lead to therapies wherein tumor cells are incapable of repairing DNA damage, therefore sensitizing the tumor cells to DNA damaging agents. Normal cells, containing intact Gl and G2 checkpoints, would still be able to repair DNA damage in the presence of the G2 checkpoint-specific inhibitor. Treatment of tumors with a radl checkpoint- specific inhibitor followed by, or along with radiation or chemotherapy would increase the efficacy of cell killing and thereby decrease the required doses of toxic DNA damaging agent

SUMMARY OF THE INVENTION In brief, the present invention provides novel radl 7 polypeptides and underlying polynucleotides The invention includes both naturally occurring and non-naturally occurring radl 7 polynucleotides and polypeptide products thereof Naturally occurring radl 7 products include distinct gene and polypeptide species within the radl 7 family, these species include those which are expressed within cells of the same animal and well as corresponding species homologs expressed in cells of other animals

Within each radl 7 species, the invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts Non-naturally occurring radl 7 products include variants of the naturally occurring products such as analogs (i.e., wherein one or more amino acids are added, substituted, or deleted) and those radl 7 products which include covalent modifications (i.e., fusion proteins, glycosylation variants, Mef'-radl7, Met^"2-Lys^''-radl7s, Gly^_1-radl7s and the like) In a preferred embodiment, the invention provides a polynucleotide comprising the sequence set forth in SEQ ID NO 1 The invention also embraces polynucleotides encoding the amino acid sequence set out in SEQ ED NO 2 A presently preferred polypeptide of the invention comprises the amino acid sequence set out in SEQ ED NO. 2

The present invention provides novel purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the human radl 7s. DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences "Synthesized," as used herein and is understood in the art, refers to purely chemical, as opposed to enzymatic, methods for producing polynucleotides "Wholly" synthesized DNA sequences are therefore produced entirely by chemical means, and "partially" synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. A preferred DNA sequence encoding a human rad 17 polypeptide is set out in SEQ ED NO. 1 The worker of skill in the art will readily appreciate that the preferred DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO 1 along with the complementary molecule (the "non-coding strand" or "complement") having a sequence deducible from the sequence of SEQ ED NO 1 according to Watson-Crick base paring rules for DNA Also preferred are polynucleotides encoding the radl 7 polypeptide of SEQ ED NO 2 The invention further embraces species, preferably mammalian and in particular mouse, homologs of the human radl 7 DNA A preferred mouse polynucleotide on the invention is set out in SEQ ID NO 3 which encodes the amino acid sequence set out in SEQ ED NO 4

The invention also embraces DNA sequences encoding radl 7 species which hybridize under highly stringent conditions to the non-coding strand, or complement, of the polynucleotide in SEQ ED NOs 1 DNA sequences encoding radl 7 polypeptides which would hybridize thereto but for the redundancy of the genetic code are contemplated by the invention Exemplary high stringency hybridization conditions are as follows hybridization at 42 °C in 5X SSPE and 50% formamide, and washing at 50°C in 0 1X SSC It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described in Ausebel, et al. (Eds ), Protocols in Molecular Biology. John Wiley & Sons (1994), pp 6 0 3 to 64 10 Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe The hybridization conditions can be calculated as described in Sambrook, et al, (Eds ), Molecular Cloning A Laboratory

Manual. Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York (1989), pp 9 47 to 9.51

Autonomously replicating recombinant expression constructions such as plasmid and viral DNA vectors incorporating radl 7 sequences are also provided Expression constructs wherein rad 17-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided

According to another aspect of the invention, host cells are provided, including procaryotic and eukaryotic cells, either stably or transiently transformed with DNA sequences of the invention in a manner which permits expression of rad 17 polypeptides of the invention Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with rad 17. Host cells of the invention are also conspicuously useful in methods for large scale production of rad 17 polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification. Knowledge of rad 17 DNA sequences allows for modification of cells to permit, or increase, expression of endogenous radl 7. Cells can be modified (e.g., by homologous recombination) to provide increased radl 7 expression by replacing, in whole or in part, the naturally occurring rad 17 promoter with all or part of a heterologous promoter so that the cells express rad 17 at higher levels. The heterologous promoter is inserted in such a manner that it is operatively-linked to rad 17 encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. 91/09955. The invention also contemplates that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the rad 17 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the rad 17 coding sequences in the cells.

The DNA sequence information provided by the present invention also makes possible the development through, e.g. homologous recombination or "knock-out" strategies [Capecchi, Science - : 1288- 1292 (1989)], of animals that fail to express functional radl7 or that express a variant of radl7. Such animals are useful as models for studying the in vivo activities of rad 17 and modulators of rad 17.

The invention also provides purified and isolated mammalian rad 17 polypeptides. Presently preferred is a human rad 17 polypeptide comprising the amino acid sequence set out in SEQ ED NO: 2. A presently preferred mouse rad 17 polypeptide is set out in SEQ ED NO: 4. Rad 17 polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Rad 17 products of the invention may be full length polypeptides. biologically active fragments, or variants thereof which retain specific rad 17 biological or immunological activity Variants, or mutants, may comprise rad 17 polypeptide analogs wherein one or more of the specified (i.e. , naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added (1) without loss of one or more of the biological activities or immunological characteristics specific for radl 7, or (2) with specific disablement of a particular biological activity of rad 17 Particularly preferred mutants of the invention comprise rad 17 polypeptides wherein regions that possess enzymatic activity have been modified Particularly preferred fragments of the invention include polypeptide regions which are conserved in human rad 17 and in species homologs to the human polypeptide. Most preferred fragments include polypeptide regions which are unique to the human radl 7 polypeptides The worker of ordinary skill in the art, through routine sequence comparison, will readily appreciate which regions of the human rad 17 polypeptide are conserved across species boundaries and which regions are unique to the human polypeptide.

Variant products of the invention include mature rad 17 products, i.e., radii products wherein leader or signal sequences are removed, having additional amino terminal residues. Rad 17 products having an additional methionine residue at position -1 (Mef'-radπ) are contemplated, as are rad 17 products having additional methionine and lysine residues at positions -2 and -1 (Met^"2-Lys^"'-radl7). Variants of these types are particularly useful for recombinant protein production in bacterial cell types

The invention also embraces rad 17 variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide such as a glutathione-S-transferase (GST) fusion product provide the desired polypeptide having an additional amino terminal residues which result from cleavage at a designed sited between the GST component and the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.

The invention further embraces rad 17 products modified to include one or more water soluble polymer attachments. Particularly preferred are rad 17 products covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the rad 17 products, or randomly attached to one or more side chains of the polypeptide.

Also comprehended by the present invention are antibodies (e.g.. monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like) and other binding proteins specific for rad 17 products or fragments thereof The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind rad 17 polypeptides exclusively (i.e., able to distinguish distinct radl7 polypeptides from the family of radl7 polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (eds), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the rad 17 polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, rad 17 polypeptides. As with antibodies that are specific for full length radl 7 polypeptides, antibodies of the invention that recognize rad 17 fragments are those which can distinguish distinct radl 7 polypeptides from the family of rad 17 polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.

Specific binding proteins can be developed using isolated or recombinant radl7 products, radl 7 variants, or cells expressing such products. Binding proteins are useful for purifying rad 17 products and detection or quantification of rad 17 products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of rad 17, especially those activities involved in signal transduction. Anti-idiotypic antibodies specific for anti-radl7 antibodies are also contemplated.

The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for rad 17 makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding rad 17 and rad 17 expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like DNA/DNA hybridization procedures carried out with DNA sequences of the invention under moderately to highly stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of radl 7, allelic variants are known in the art to include structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to radl 7 Similarly, non-human species genes encoding proteins homologous to rad 17 can also be identified by Southern and/or PCR analysis and useful in animal models for radl7-related disorders As an alternative, complementation studies can be useful for identifying other human radl 7 products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of rad 17

Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express rad 17 Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a rad 17 locus that underlies a disease state or states, including cancer (i.e., bladder, head and neck, cancers as well as small cell lung tumors), immune and proliferative disorders, cirrhosis, and rheumatoid arthritis.

Also made available by the invention are anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding rad 17. Full length and fragment anti-sense polynucleotides are provided The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to rad 17 RNA (as determined by sequence comparison of DNA encoding rad 17 to DNA encoding other known molecules) as well as (ii) those which recognize and hybridize to RNA encoding other members of the rad 17 family of proteins. Antisense polynucleotides that hybridize to multiple DNA encoding other members of the rad 17 family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of rad 17 by those cells expressing rad 17 mRNA. The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of radl 7s DNA and amino acid sequence information for radl 7 also permits identification of binding partner compounds with which a rad 17 polypeptide or polynucleotide will interact. Agents that modulate (i.e., increase, decrease, or block) rad 17 activity or expression may be identified by incubating a putative modulator with a rad 17 polypeptide or polynucleotide and determining the effect of the putative modulator on rad 17 activity or expression. The selectivity of a compound that modulates the activity of the radl 7 can be evaluated by comparing its binding activity on rad 17 to its activity on other rad 17 enzymes. Cell based methods, such as di-hybrid assays to identify DNAs encoding binding compounds and split hybrid assays to identify inhibitors of rad 17 polypeptide interaction with a known binding polypeptide, as well as in vitro methods, including assays wherein a rad 17 polypeptide, rad 17 polynucleotide, or a binding partner are immobilized, and solution assays are contemplated by the invention.

Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to a rad 17 polypeptide or a radl7-encoding nucleic acid, oligonucleotides which specifically bind to a rad 17 polypeptide or a rad 17 gene sequence, and other non-peptide compounds (e.g., isolated or synthetic organic and inorganic molecules) which specifically react with a rad 17 polypeptide or underlying nucleic acid. Mutant rad 17 polypeptides which affect the enzymatic activity or cellular localization of the wild-type rad 17 polypeptides are also contemplated by the invention. Preferred mutants of the invention include those which result in loss of ATPase activity, as well as mutants wherein modifications are effected in regions found to be conserved in mammalian and yeast rad 17 polypeptides as discussed above. Presently preferred targets for the development of selective modulators include, for example: (1) regions of the rad 17 polypeptide which contact other proteins and/or localize the rad 17 polypeptide within a cell, (2) regions of the rad 17 polypeptide which bind and/or hydrolyze substrate, (3) allosteric cyclic nucleotide-binding site(s) of the rad 17 polypeptide, (4) phosphorylation site(s) of the rad 17 polypeptide, (5) regions of the rad 17 polypeptide which are involved in multimerization of rad 17 subunits, (6) regions of the rad 17 polypeptide which contact polynucleotides, and (7) regions of the rad 17 polypeptide which possess enzymatic activity. Still other selective modulators include those that recognize specific rad 17 encoding and regulatory polynucleotide sequences. Modulators of rad 17 activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which rad 17 activity is known, believed, or proposed to be involved as discussed herein

The present invention further embraces screening assays to identify modulators of rad 17 enzymatic activities The presently preferred screening assay of the invention embraces identification of modulators of rad 177 ATPase enzymatic activity Assays of the invention include those comprising the steps of contacting rad 17 and a substrate in the presence and absence of a compound, determining binding between rad 17 and the substrate in the presence and absence of the compound, identifying the compound as a modulator of radl7/substrate binding wherein a difference in radl 7/substrate binding is observed in the presence of the compound In assays of the invention, detecting increased binding in the presence of the compound indicates the compound is an activator and instances wherein decreased rad 17/substrate binding is detected, the compound is identified as an inhibitor Assays of the invention include those wherein (i) one of rad 17 and the substrate is immobilized, (ii) the other is detectably labeled, (iii) the substrate and rad 17 are contacted in the presence and absence of a compound, and (iv) the ability of the compound to modulate radl 7/substrate binding is assessed by a change in the amount of immobilized label compared to the amount of immobilized label in the absence of the compound The invention further embraces solution assays wherein either binding partner rad 17 and/or the substrate is detectably labeled, the binding partners are contacted in the presence and absence of a compound, binding between rad 17 and the substrate is detected in the presence and absence of the compound, and the compound is identified as a modulator of binding if a change in radl 7/substrate binding is detected

The invention contemplates that mutations in the rad 17 gene that result in loss of normal function of the rad 17 gene product underlie human disease states in which failure of the cell cycle checkpoint is involved. Gene therapy to restore rad 17 activity would thus be indicated in treating those disease states (for example, various forms of cancer described herein). Delivery of a functional rad 17 gene to appropriate cells is effected in vivo or ex vivo by use of vectors, and more particularly viral vectors (e.g. , adenovinis, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g. , liposomes or chemical treatments). For reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma,

Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it is contemplated that in other human disease states preventing the expression of or inhibiting the activity of rad 17 will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of radl 7. Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to rad 17 expression control sequences or rad 17 RNA are introduced into cells (e.g. , by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the rad 17 target sequence in the cell and prevents transcription or translation of the target sequence. Phosphothioate and methylphosphate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.

Moreover, for example, if a particular form of cancer results from a mutation in a gene other than radl 7, an agent which inhibits the transcription or the enzymatic activity of rad 17 and thus the cell cycle checkpoint may be used to render cancerous cells more sensitive to chemotherapy or radiation therapy. The therapeutic value of such an agent lies in the fact that current radiation therapy or chemotherapy in most cases does nothing to overcome the ability of the cancerous cell to sense and correct the DNA damage imposed as a result of the treatment. As a result, a cancer cell can simply repair the DNA damage. Modulating agents of the invention may therefore be chemotherapy and radiation adjuvants or may be directly active as chemotherapeutic drugs themselves.

DETAILED DESCRIPTION The present invention is illustrated by the following examples. Example

1 details the identification, cloning, and sequence analysis of the cDNAs encoding the human and mouse Rad 17 proteins Example 2 describes the tissue expression pattern of human Rad 17 (hRadl7) mRNA Example 3 details the expression and purification of recombinant hRadl7 and hRad!7-mutant fusion proteins from bacteria Example 4 describes the immunological reagents generated for Rad 17 detection and purification.

The chromosomal localization of the hRadl 7 gene is described in Example 5 Expression of recombinant hRadl7 in mammalian cell lines, isolation of stably transfected cell lines, and cellular localization of a fluorescent hRadl7 fusion protein is described in Example 6. Example 7 details a biochemical assay for the analysis of hRadl7 ATPase activity. Example 8 describes experiments designed to identify proteins that associate with hRadl7.

Unless otherwise indicated, small scale plasmid preparations (minipreps) were a carried out with Wizard Plus kit (Promega), large scale plasmid preparations performed with Midi-Prep or Maxi-Prep kits (Qiagen), DNA purification from electrophoresis gels was carried out using Gene Clean II kit (BiolOl), and all restriction digests and ligation reactions were carried out according to manufacturer's guidelines.

Example 1 Cloning of Human Radl7 cDNA

In an attempt to identify a human homolog of S.pombe rad 17, the polynucleotide sequence of rad 17 was utilized as the query sequence in a search of the

National Center for Biotechnology Information (NCBI) expressed sequence tag (EST) database. The EST database provides 5 ^' and/or 3 ' nucleotide sequences for cDN A clones from various libraries, and for each EST, the cDNA clone source is also identified. The TBLASTn program available from NCBI was used for nucleotide sequence comparison and ESTs having significant homology to the yeast protein were assembled using

Sequencher 3.0 DNA analysis software (Gene Codes Corp.) in order to identify source clones including the largest open reading frame.

Three human cDNA clones were identified by their 5^' and/or 3 ' sequences in the EST database as being homologous to the yeast query sequence: (i) 5' EST AA133547 (SEQ ID NO: 5) indicated radl7 homology with clone 586844; (ii) 3' EST

AA287094 (SEQ ID NO: 6) indicated radl 7 homology with clone 701704; and (iii) 3 ' EST H20056 (SEQ ED NO: 7) indicated radl7 homology with clone 172809. Clone 586844 was found to include the largest cDNA insert of the three human cDNAs. The search also identified two mouse clones: (i) 5' EST AA109778 (SEQ ID NO: 8) indicated radl7 homology with clone 519966 and (ii) 5' EST AA645646 (SEQ ID NO: 9) indicated rad 17 homology with clone 1020744. The five clones identified were obtained (I.M.A.G.E.) and two human clones, 586844 and 701704, and one mouse clone, 519966, were sequenced with primers that hybridized to the vector DNA and primers designed to hybridize to the cDNA. The nucleotide sequence for human clone 586844 is set out in SEQ ID NO: 10, human clone 701704 is set out in SEQ ID NO: 1 1 and the sequence for mouse clone 519966 is set out in SEQ ID NO: 3. Mouse clone 519966 was found to include an incomplete reading frame encoding 293 amino acids which corresponded to the carboxy terminus of the yeast rad 17 protein. The deduced amino acid sequence for the mouse clone is set out in SEQ ED NO: 4.

Sequence analysis of the human clones revealed several difference which led to several proposed modifications in order to derive a more complete nucleotide sequence for the putative human radl7. The 5' 132 nucleotides in 586844 were determined to be part of an unrelated cDNA as indicated by homology of the region with an EST in the database otherwise having no homology to the query sequence. The extraneous 132 nucleotides were therefore deleted from the predicted reading frame. Upon considering the deletion, it was determined that the longer 586844 cDNA encoded an incomplete reading frame as evidenced by the absence of an initiating methionine codon.

In comparison of sequences for 586844 and 701704, is was found that 701704 included a 1 12 nucleotide sequence not found in 586844 and that the 112 nucleotide deletion in the 586844 cDNA resulted in a frameshift in the predicted open reading frame of the clone that resulted in a region of the deduced amino acid sequence with little or no homology to the yeast protein. The absence of the 112 bp sequence from the 586844 cDNA may have resulted from alternate splicing of the hRadl7 mRNA (i.e., selection of a 3^' splice site 112 nucleotides downstream of the correct site). In fact, DNA sequences preceding the 112 bp sequence (AG) and at the end of the 112 bp sequence

(pyrimidine-rich sequence followed by AAAAG) showed a high resemblance to the consensus sequence for exon intron and intron/exon junctions, respectively [Lewin GENES IV. Oxford University Press: New York (1997), p.88]. The most common sequence at the 3 ^' end of an exon is AG, and at the 3 ' end of an intron is C AG preceded by a stretch of pyrimidines. It is possible that an intron is present after nucleotide 1019 of 586844 and PCR analysis of genomic DNA is used to verify this prediction. However, in view of the observation that two other clones in the EST database overlapped the predicted 586844 reading frame and also contained the 112 bp sequence, the deletion was restored in the predicted 586844 reading frame. With the deletion restored, the amino acid sequence encoded by 586844 differed from that of 701704 at two residues; in 586844, residues at position 127 and 281 were proline and leucine, respectively, and in 701704, amino acids at the same relative positions were histidine and proline. Because the corresponding amino acids at both positions in S.pombe rad 17 are proline, nucleotides in 586844 encoding leucine at position 281 were modified to encode proline.

Finally, the 5^' EST of 701704 was found to be incorrect in the database; the true 5' region was identical to a sequence identified as the 5^' sequence of another clone, 701702, and, following purchase of the 701702 cDNA and sequence analysis, it was found that the clone it did not contain a cDNA insert. The 5^' EST of 701704 reported in the database was therefore removed from consideration as part of the human sequence. In order to physically construct a polynucleotide encoding a human rad 17 species homolog, the 586844 cDNA was modified to insert the 1 12 bp region (including flanking nucleotides which encode proline at position 281) from 701704 using a technique wherein an internal 586844 fragment was replaced with a corresponding sequence from 701704. The ligation reaction joined three sequences derived from either 586844 or 701704: a XhoVHindm 5^' 586844 fragment of approximately 3.6 kb, a 510 bp

HindllUHaem fragment from 701704, and a 1522 bp HaelTVXhol 3 ^' fragment from 586844. Prior to ligation, all fragments were purified from agarose gels (BiolOl) and the resulting ligation product was designated pAR17-l (assembled hRadU). The polynucleotide and predicted amino acid sequences for pAR17-l are set out in SEQ ED NOs: 12 and 13, respectively.

The ligation mixture was transformed into E.coli TOPI OF' cells (Invitrogen) and transformants were selected on LBM agar containing 100 μg/ml carbenicillin. Restriction digests were performed on small scale plasmid preps to identify colonies having a pARl 7-1 -encoding plasmid. In order to clone the 5' sequence including an initiating methionine codon of hRadl7 cDNA, RACE PCR was employed as follows. PCR primers, RAD17.GSP1 (SEQ ID NO: 14) and RAD17.NGSP1 (SEQ ID NO: 15), were synthesized based on gene sequences for use in combination with anchored primers, API (SEQ ED NO 16) and AP2 (SEQ ED NO 17) that hybridized to the 5 ^' ends of human testis Marathon Ready cDNA (Clontech) A first amplification was carried out using 5 μl Marathon Ready cDNA in a 50 μl reaction containing 0 2 μM primers AP I and RAD 17 GSP1, 200 μM dNTPs, IX Perkin Elmer buffer with MgCl₂, and 5 units AmpliTaq polymerase (Perkin Elmer)

The reaction mixture was first heated for one minute at 94°C, then subjected to the following conditions, (i) five thermocycles of 94°C for 30 seconds and 72°C for three minutes, (ii) five thermocycles of 94 °C for 30 seconds and 70 °C for three minutes, (iii) twenty-five thermocycles of 94 °C for 30 seconds and 68 °C for four minutes, and (iv) a final incubation for five minutes at 72 °C One microliter from the resulting reaction was used as template for a second round of amplification with internal primers AP2 and RAD17.NGSP1 The amplification reaction was performed in a 50 μl total volume with 0.2 μM each primer, 200 μM dNTPs, IX KlenTaq PCR buffer (Clontech), and 1 μl KlenTaq polymerase mix (Clontech). Reaction mix was subjected to a first heating step at 94°C for one minute followed by thirty thermocycles of 94°C for 30 seconds and 68°C for three minutes followed by a final incubation at 72 °C for five minutes An aliquot of the reaction mixture was analyzed on a 1 5% agarose gel and amplification products of approximately 80 to 800 bp were detected The PCR products were inserted into vector pCR3.1 using the Eukaryotic TA Cloning Kit (Invitrogen) and the resulting ligation mixture transformed into E.coli TOPI OF cells (Invitrogen). Transformants were selected on LBM agar containing 100 μg/ml carbenicillin and colonies were picked for use as template in PCR reactions to screen for clones with the largest inserts.

PCR reactions (10 μl total volume) included cells transferred with a toothpick, 0.2 μM primers AP2 and RAD17.NGSP1, 200 μM dNTPs, IX Perkin Elmer buffer with MgC^, and 1 unit AmpliTaq (Perkin Elmer). Amplification was carried out with a first step of heating for 30 seconds at 94°C, followed by 30 thermocycles of denaturation at 94 °C for 20 seconds, annealing at 65 °C for 30 seconds, and extension at 72°C for one minute, followed by a final incubation for 5 minutes at 72°C. PCR products were analyzed on a 2% agarose gel. Two clones with the largest inserts (approximately 300 bp) were chosen for DNA preparation and DNA sequence analysis. Inserts in both clones contained cDNA overlapping the 5^' region of clone 586844 and containing 11 bp of additional 5' sequence that included an ATG sequence The ATG sequence was in reading frame with the predicted coding region for clone 586844 and was presumed to represent the initiation codon of the hRadl7 gene based on comparison with amino terminal regions of other Rad 17-related yeast proteins It is possible, however, that additional 5 translated sequences of the putative hRadl7 exist in view of the fact that the cloned sequence surrounding the proposed start codon is not extensive enough to permit strong conclusions regarding the degree of homology in the region with consensus eucaryotic translation initiation sites [Kozak, J. Mol Biol 196:941-950 (1987) ]

Assembly of pAR17-l cDNA with the 1 1 bp of additional 5 ^' sequence resulted in a hRadl7 cDNA that was 2526 nucleotides in length, including a four bp 5^' untranslated region, a 2010 nucleotide open reading frame (encoding a 670 amino acid polypeptide) and a 512 nucleotide 3 ^' untranslated region The hRadl 7 protein sequence also contained an ATP -binding motif, known as the Walker A box or P-loop motif, that is highly conserved in many different ATP-hydrolyzing enzymes in eukaryotes and prokaryotes [Yoshida and Amano, F.E.B.S. Letts. 359.1-5 (1995)] The polynucleotide and amino acid sequences for the resulting modified hRadl7 cDNA are set out in SEQ ID NO 1 and 2, respectively

The deduced carboxy terminal sequences of the predicted human and mouse Radl 7 proteins were 85% identical to each other over a 285 amino acid region, taking into consideration three gaps of one to six residues each Comparison of the hRadl7 sequence with the mouse 1020744 clone revealed 81% identity (without gaps) over a 154 amino acid region near the amino terminus of the mouse clone

Comparison of the 670 amino acid hRadl7 sequence with known proteins using NCBI BLASTp analysis with default search parameters revealed that the amino acid sequence was most closely related to S.pombe rad 17 and S.cerevisiae RAD24. S.pombe rad 17 is approximately 42% identical to hRadl7 over six non-overlapping regions that average 39 amino acids in length. S. cerevisiae RAD24 is approximately 52% identical to hRadl7 over five non-overlapping regions that average 24 amino acids in length

Example 2

Northern Analysis

In order to determine the expression pattern of hRadl7, multiple tissue and human cell line Northern blots (Clontech) were probed with portion of the hRadl 7 cDNA. A 294 bp fragment was amplified by PCR in a reaction including α^P-dCTP and α³²P-dTTP with using primers RAD17.7 (SEQ ID NO: 18) and RAD17.8 (SEQ ID NO 19) and 100 ng pAR17-l as template DNA. Reaction conditions included IX Perkin Elmer buffer with MgCl₂, 250 μM dATP and dGTP, 100 μCi radiolabled dCTP and dTTP, and 10 μM unlabeled dCTP and dTTP The PCR mixture was first incubated at 94 °C for eight minutes, followed by forty thermocycles of denaturation at 94 °C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 30 seconds. Blots were probed according to manufacturer's (Clontech) suggested protocol and subjected to autoradiography.

Results from the tissue blot indicated a transcript size of approximately 3000 nucleotides which was consistent with the size of the cDNA identified in Example 1. The hRadl7 mRNA was highly abundant in testis, but present at low levels in all other tissues tested (including spleen, thymus, prostate, ovary, small intestine, colon, leukocyte, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas). The result that hRad!7 was expressed in all tissues tested, albeit at varying levels, was expected in that expression in all cells would be presumed for a general cell cycle checkpoint gene. It is possible that the high level of expression in testis indicates that hRadl7 participates in meiotic recombination. A similar expression pattern has been observed with ATR, another human cell cycle checkpoint gene.

In all probed cell lines (HL-60, HeLa S3, K562, MOLT-4, Raji, SW480, A549, and G361), hRadl7 mRNA of approximately 3000 nucleotides was detected.

Example 3 Expression of hRad 17

Construction of a hRad 17 Expression Vector

In order to attempt expression of the hRad 17 polypeptide, the coding sequence from pAR17-l was inserted into a bacterial expression vector designed to produce the desired protein as a glutathione-S-transferase (GST) fusion as described below.

The hRadl7 cDNA in pAR17-l (as set out in SEQ ID NO: 12 and without the 11 bp sequence identified by RACE PCR) was modified at the 5^' end by PCR to generate a start codon and convenient restriction sites for cloning into the GST gene fusion expression vector, pGEX-5X-l (Pharmacia Biotech) The resulting cDNA fragment was termed amR17 because it was assembled from two EST clones (586844 and 701704) and was modified at the 5' end to create restriction sites and eliminate 149 5 ^' nucleotide from the 586844 insert (eliminating codons 1 through 8)

Generation of the GST-amR17 expression plasmid, pGEX-amR17, was performed as follows A PCR primer, RAD 17 5 ^'MOD (SEQ ED NO 20), was designed in order to modify the amplification product to include convenient cloning sites, i.e., restriction sites for EcoRI, BamH , and Ncol) PCR was carried out in a 50 μl volume reaction containing 40 ng pAR17-l template DΝA, 400 ng each RAD 17 5'MOD, 3 units of Taq DΝA polymerase (AmpliTaq, Perkin Elmer), IX Perkin Elmer buffer with MgC , and 0 2 mM dΝTPs The mixture was first heated at 94 °C for two minutes and subjected to ten thermocycles (94°C for 20 seconds, 48°C for 30 seconds, 72°C for 50 seconds) after which 270 ng of primer RAD 17 GSP 1 was added The resulting mixture was subjected to 30 thermocycles (94°C for 20 seconds, 62°C for 30 seconds, 72°C for 50 seconds) followed by a five minute incubation at 72 °C. The PCR product was purified with Gene Clean II (Bio 101), digested with EcoRI and H/ndlll (to provide a fragment of approximately 505 bp), and purified from an agarose gel

The pGΕX-5X-l vector was digested with EcoRI and Xhol and purified from an agarose gel. Similarly, pAR17-l was digested with Hindlll and Xhol and the approximately 2 kb hRad 17 fragment also purified from an agarose gel The two fragments were combined with the EcoRI Hwdlll PCR product described above in a ligation reaction that produced pGΕX-amRl 7 The ligation products were transformed into competent Exoli TOPI OF' cells (Invitrogen) according to manufacture's suggested protocol and transformants were selected on LBM agar containing carbenicillin Plasmid preparations were analyzed by restriction digestion and DΝA sequence analysis to verify the correct assembly of pGΕX-amR17 The polynucleotide and amino acid sequences for amR17 are set out in SEQ ED ΝOs 31 and 32, respectively.

Construction of an Expression Vector Encoding a hRad 17 Mutant

In order to create a hRad 17 mutant with defective ATPase function, the nucleotide sequence encoding lysine (AAG, nucleotides 398 to 400 in hRad7 cDΝA (including the 5' RACE product) in the Walker A box was mutated to encode glutamic acid (GAG). Mutation of the lysine residue in other proteins has been shown to abolish or severely depress the activity of ATPases [Senior, et al, J. Biol Chem. 2(55:6989- 6994(1993)]. In order to generate the mutation, two oppositely oriented and complementary mutagenic PCR primers, RAD17.KE (SEQ ED NO: 21) and

RAD17.KEbot (SEQ ED NO: 22) were synthesized. A first series of amplifications was carried out with each of the primers in combination with flanking primers and the resulting amplification products were then used as templates in a subsequent PCR reaction including only the flanking primers to amplify the lysine-to-glutamate mutant gene fragment.

The first PCR reactions were performed in 20 μl mixture containing a 10 ng pGEX-amR17 template DNA, 100 ng each primer (either RAD17.KE with RAD17.GSP1 or RAD17.KEbot with RAD17.5'MOD), 1.5 units of Taq DNA polymerase (AmpliTaq, Perkin Elmer), IX Perkin Elmer buffer with MgCl₂, and 0.2 mM dNTPs. The reaction mixtures were heated at 94 °C for 30 seconds and subjected to 25 thermocycles (94°C for 20 seconds, 50°C for 30 seconds, 72°C for 50 seconds), followed by a five minute incubation at 72 °C. The 173 bp product from the reaction using the RAD17.KE and RAD17.GSP1 primers and the 402 bp amplification product from the reaction using the RAD17.KEbot and RAD17.5'MOD primers were purified using a 2% agarose gel as follows.

The fragments in gel slices were frozen at -70 °C, thawed, and centrifuged for five minutes in an Eppendorf microfuge. One microliter of each supernatant was combined and PCR carried out with the RADπ^TvlOD and RAD17.GSP1 primers using the above conditions. The resulting 538 bp PCR product was purified from a 2% agarose gel, digested with EcoRI and Hwdlll, and ligated to the large EcoRI/H/^'wdlll fragment of pGΕX-amR17 previously gel-purified. The ligated products was transformed into competent E.coli TOPI OF¹ cells (Invitrogen) according to manufacturer's suggested protocol and transformants were selected on LBM agar containing carbenicillin. Plasmid preparations from selected colonies were analyzed using restriction digestion and sequence analysis to verify correct assembly and inclusion of the desired point mutation ofpGEX-amR17.KE Protein Expression and Purification

The GST-amR17 fusion protein was first purified on a glutathione matrix as described below The GST protein (from parental vector pGEX-5X-l) was purified by this same method A culture of pGEX-amR17 (in TOPI OF' cells) was grown at 30 °C in LBM with 0 1 mg/ml carbenicillin to an absorbance of 0.5-0.7 at 600 nm EPTG was then added to a final concentration of 1 mM to induce gene expression and after four hours continued growth, the cells were centrifuged at 10,000 x g for five minutes Cell pellets were stored at -70°C until use Thawed pellets were resuspended in Lysis Buffer containing 25 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.1% NP-40, 150 mM NaCl, 5 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin, 5 μg/ml aprotinin Cells were lysed by sonication and centrifuged 20 minutes at 10,000 x g The supernatant was mixed with l/50th volume of reduced glutathione (GSH) agarose (Sigma) for 2.5 hours at 4°C The GSH agarose was sedimented by low speed centrifugation and the pellet was washed four times with 33 volumes of Wash Buffer containing 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.1% NP-40, 150 mM NaCl, 0.2 mM PMSF, and 1 μg/ml aprotinin. Proteins were eluted from the glutathione matrix with 3.3 volumes of 20 mM Glutathione Buffer containing 120 mM NaCl and 50 mM Tris-HCl, pH 9 5 Elution was carried out for 10 minutes at 0°C and the slurry was drained through a PolyPrep column (BioRad) to remove the matrix. The eluate was dialyzed at 4°C against storage buffer containing 20 mM Tris-HCl, pH 8, 0 1 mM EDTA, 10% glycerol, 50 mM KC1, and 1 mM DTT and protein concentration was determined with Pierce Coomassie Plus with BSA as a standard. Protein was stored in aliquots at -70 °C after freezing in liquid nitrogen

Analysis of the GST-amR17 protein on Coomassie stained gels revealed that the full-length fusion protein (approximately 105 kDa) only accounted for about 10% of the total protein. The remainder if the protein ranged in size from about 28 to 80 kDa

Western blot analysis with anti-hRadl7 peptide antisera (described below in Example 4) and with antibodies that recognized GST verified that the 105 kDa protein included both hRad 17 and GST epitopes. Similar antibodies which recognize GST domains are commercially available from, for example, Pharmacia Biotech. All the smaller proteins appeared to be truncations of the fusion protein containing GST and different lengths of the hRad 17 protein.

Various expression and purification conditions were tested in order to ~n . optimize the production of the full-length GST-amR17. It was determined that in the absence of induction (no IPTG) the basal level of GST-amR17 expression contained proportionately more full length protein compared to the smaller breakdown products Additionally, the smaller products appeared to elute from Q Sepharose (Pharmacia) in lower salt than the full-length GST-amR17. Additionally, the expression vectors were transformed into E. coh strain BL21 known to be deficient in at least one protease gene which may have accounted for the high level of truncated expression products.

BL21 cells transformed with either pGΕX-amR17 or pGEX-amR17.KE were grown to A₆₀₀ of 1 to 2 absorbance units and centrifuged at 10,000 x g for 10 minutes. The cell pellet was frozen at -70°C, thawed, and resuspended in Lysis Buffer

2 containing 25 mM Tris-HCl, pH 7 5, 10 mM EDTA, 0.4% NP-40. 150 mM NaCl, 5 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin, and 5 μg/ml aprotinin The cells were sonicated with a macro-tip at amplitude 50 (three times for 30 seconds) in 50 ml aliquots and cell debris was removed by centrifugation (10,000 x g for 10 minutes). The supernatant was diluted with three volumes of Dilution Buffer containing 25 mM Tris-

HCl, pH 7.5, 5 mM EDTA, 1 mM PMSF, 0.5 μg/ml leupeptin, and 2.5 μg/ml aprotinin, and filtered through a 0.45 micron cellulose acetate membrane. The filtrate was loaded onto a Q Sepharose FF (Pharmacia) column at a rate of 300 ml/cm² per hour. The column was washed with two bed volumes of 40 mM NaCl in Buffer Q containing 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0 1% NP-40, 1 mM DTT, 0.2 mM PMSF, 2.5 μg/ml aprotinin, and 0.5 μg/ml leupeptin. A linear gradient (four bed volumes) of 40 to 600 mM NaCl in Buffer Q was run over the column, and 0.2 column volume fractions were collected. Presence of the 105 kDa GST-amR17 (and GST-amR17.KE) was assessed on Western blots. Fractions eluting between 350 to 450 mM NaCl were found to be enriched for the full-length protein and were pooled. The pooled fractions were diluted with Buffer Q to reduce the NaCl concentration to approximately 200 mM and the proteins were mixed with l/20th volume of GSH agarose over night (approximately 15 hours) at 4°C. The slurry was poured into a PolyPrep column (BioRad) and drained, after which the GSH matrix was washed with 10 bed volumes Wash Buffer (see above), and eluted with 20 mM Glutathione Buffer (see above). Eluates were dialyzed against Dialysis Buffer 2 (20 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, 5 mM β-mercaptoethanol, 50 mM KC1, and 10% glycerol) and stored at -70°C following freezing in liquid nitrogen. Analysis of the proteins on Coomassie stained gels revealed that the preparation yielded a greater proportion of full-length GST-amR17 than the previous method. Overall yield of GST-fusion protein from uninduced cells, however, was low.

Example 4 Production of hRAD17 Antibodies Recombinant GST-amR17 and synthetic peptides were used as antigens to raise antibodies to hRad 17 in rabbits and mice as follows.

The hRad 17 peptide sequence (SEQ ID NO: 2 as deduced from EST 586844 and 701704 sequences) was provided to Quality Controlled Biochemicals, Inc. (QCB, Hopkinton, MA) which was contracted to identify likely immunogenic peptide sequences, synthesize the peptides, and immunized rabbits for the production of antisera.

It was realized later that the peptide sequence provided to QCB was incorrect, so the two peptide antigens designed by QCB did not match the hRad 17 sequence. Synthetic peptide #1 (acetyl-KRKLKEVETWLKAQVLC-amide) was identical the hRad 17 sequence at positions 1, 3 and 6 through 16 (corresponding to residues 96, 98, and 101 through 1 1 1 in Radl 7), and contained a carboxy terminal cysteine residue to aid in cross-linking of the peptide to a solid matrix. Synthetic peptide #2 (acetyl-CSGDNNQKLLFPKEIQEE- amide) was identical to hRad 17 sequence (residues 252 through 268) except at position 8 (hRadl7 residue 258) and at the amino terminal cysteine.

Anti-peptide polyclonal sera from two rabbits (injected with both peptides) was found to recognize GST-amR17 on Western blots. Specificity of the antisera was determined by the observation that detection of GST-amR17 could be prevented by pre- incubating the synthetic peptides with the antisera before probing.

The two antigenic peptides were covalently cross-linked to a solid matrix (at QCB) and were used to purify peptide-specific antibodies from the rabbit polyclonal sera. Western blot analysis determined that both of the affinity-purified antibody preparations recognized GST-amR17.

In a second approach to antibody production, R & R Rabbitry (Stanwood, WA) was contracted to immunize rabbits with GST-amR17. Polyclonal antisera from the immunized rabbits was demonstrated to recognize GST-amR17, GFP-Radl 7 (discussed below), and an human endogenous protein of about 75 kDa (the predicted size of hRad 17). In a third approach to antibody production, GST-amRl 7 and the synthetic peptides #1 and #2 described above were used to immunize mice. Mice were subcutaneously injected with 40 μg GST-amRl 7 ( or 50 μg specific peptide) in Complete Freund's Adjuvent (CFA) and booted every three weeks with 20 μg GST-amRl 7 (or 50 μg specific peptide) in Incomplete Freund's Adjuvant. Peptides were crosslinked to KHL using a kit (Pierce) according to manufacturers 's suggest protocol. All antisera were determined to be im unoreactive with recombinant hRad 17 in ELISA and Western blot assays.

One mouse immunized with peptide #1 was chosen for production of hybridoma cell lines. Two pre-fusion boosts with 40 μg GST-amRl 7 in calcium-, magnesium-free PBS (CMF-PBS) were performed four and five days prior to the fusion protocol. Hybridomas were generated according to previously published protocols [Harlow, et al, supra, Chapter 6]. Two isolated hybridomas, designated 282E and 282G, were cloned that were reactive against peptide #1 in an ELISA assay, and recognized GST-amRl 7 on Western blots.

Example 5 Chromosome Mapping of hRad 17

In order to map the chromosomal location of genomic hRad 17 sequence, the following protocol was carried out. Briefly, PCR was carried out on pools of genomic DNA including a single chromosome. After identification of a positive pool, PCR was carried out on DNA comprising fragments of chromosomal DNA derived from the identified first identified pool. Results from the second round of amplification were submitted for analysis after which a chromosomal locus was identified. Gene-specific oligonucleotides were used in PCR reactions with human, mouse, and hamster genomic DNA to identify primer sets that would amplify specific human gene fragments. Subsequent PCR reactions were performed with a human/rodent somatic cell hybrid mapping panel #2 (Coriell Cell Repositories) to localize the PCR fragments to specific human chromosomes Fine mapping was performed by PCR with the Stanford G3 Radiation Hybrid (G3/RH) DNA panel (Research Genetics) DNA sequence analysis distinguished the hRadl 7-derived PCR products from those derived from pseudo-genes or Radl 7 gene family members

PCR reactions on the entire somatic cell hybrid panel were performed with two non-overlapping primer sets, RAD17 6 (SEQ ED NO 233) with RAD17 PSGN (SEQ ED NO 24) or RAD 17.4-2 (SEQ ED NO 25) with RAD 17 7 (SEQ ID NO 18) Reaction mixtures (20μl) included 100 ng of each primer pair, 200 μM dNTPs, IX Perkin Elmer Taq polymerase Buffer, IX Perkin Elmer MgCl₂,

1 μl (approximately 300 ng) of somatic cell hybrid DNA or control DNA (human, mouse, and hamster), and 2 units Taq DNA polymerase (AmpliTaq, Perkin Elmer) Reaction conditions included an initial incubation at 94 °C for 30 seconds followed by 35 thermocycles (94°C for 20 sec , 62°C for 30 sec, 72°C for 30 sec). After a final extension step for five minutes at 72 °C, the samples were analyzed by electrophoresis on 10%) polyacrylamide gels

With the primer set of RAD 17 6 and RAD 17 PSGN, a 242 bp product was detected from hRad 17 cDN A, human genomic DNA (gDN A), and human chromosome 7-containing DNA With the primer set of RAD17 4-2 and RAD17.7, a 152 bp product was amplified from hRad 17 cDNA, human gDNA, and human chromosome 13-containing DNA Additionally, this latter primer set amplified a product about 100 bp larger from human gDNA and chromosome 5-containing DNA

A third and fourth primer set were used in PCR reactions with chromosome 5, 7, and 13 somatic cell hybrid DNA (and cDNA and gDNA controls) Reaction conditions were as described above, except with the third primer set,

RAD 17.6 (SEQ ED NO. 23) with 586844.1 (SEQ ED NO 26), a hybridization temperature of 50 °C was used, and with the fourth primer set, RAD 17.10 (SEQ ID NO: 27) with RAD17.1 1(SEQ ID NO: 28), a hybridization temperature of 54 °C was used. The products were analyzed as above The RAD 17 6 and 586844.1 primers amplified a 129 bp product from the cDNA, gDNA, chromosome 7 and chromosome 13 DNAs Primers RAD 17.10 and RAD 17.1 1 amplified a 186 bp product from the cDNA, gDNA, chromosome 5 DNA

The above chromosome-specific amplification products were purified from the polyacrylamide gels and sequence analysis of the DNAs revealed the following The chromosome 7 products included many base pair differences from the cDNA, the larger of which, produced using primers RAD 17.6 and RAD17 PSGN, included 14 base changes and a one base insertion (resulting in nine codon changes, including a termination codon before the frameshift) From this observation, it was determined that a pseudogene of Rad 17 lies on chromosome 7. The two non- overlapping fragments amplified from chromosome 13 included several base differences from the cDNA, but no frameshifts or termination codons were introduced

The product from using primer RAD 17 4-2 and RAD 17 7 with chromosome 13 had twelve bp differences and a three bp deletion (resulting in eight codon changes) The amplification product from primers RAD 17 6 and 586844 1 with chromosome 13 included 12 bp changes, resulting in nine codon changes (including a glycine-to-serine change at the first conserved glycine of the Walker A box) From this observation, it was predicted that chromosome 13 harbors a Rad 17 pseudogene or gene family member The PCR products using chromosome 5 exactly matched the hRad 17 cDNA sequence except for an 84 bp putative intron in the product using primers RAD 17.4-2 and RAD 17.7 product. From this observation, it was concluded that hRad 17 is encoded on chromosome 5

A radiation hybrid panel created at the Stanford Human Genome Center (G3/RH, purchased from Research Genetics) was screened by PCR using the primers RAD17.7 and RAD17.4-2 Reaction conditions were as described above, except 100 ng G3/RH DNAs served as the templates Products were analyzed on 10% polyacrylamide gels and scored for presence of the 152 bp product identified above using chromosome 13 and the 236 bp product identified using chromosome 5 Scores of the 83 G3/RH DNAs were submitted to the RH server at Stanford (http://www-shgc stanford.edu/RH/rhserver_forrn2.html). By analyzing the map locations of the closely linked STS DNAs and their flanking markers, it was deduced that amf203xal mapped to 13ql4 and SHGC- 14007 maps to 5q 13.1, a region reportedly to be mutated in several tumor cell lines [Carey, et al, Anticancer Res. 73.2561-2568 (1993), Van Dyke et al, Genes, Chromosomes and Cancer 9.192-206 (1994)].

Example 6 Expression of hRadl7 in Mammalian Cells Construction of Expression Plasmids

The hRad 17 gene was cloned into vectors for inducible expression in mammalian cell lines. The vectors were designed to express two epitope-tagged hRad 17 fusion proteins designated GFP-hRadl7 and FH-hRadl7 (called GFP-R17amf and FH-R17amf below). GFP-hRadl7 encoded hRad 17 as a fusion protein with the Green Lantern Green Fluorescent Protein (GFP) which allows microscopic detection of the fluorescent protein within the cell under ultraviolet light. FH-hRadl7 encoded hRad 17 as a fusion protein with the FLAG epitope (to permit immuno-detection and immuno-purification) and an epitope consisting of six consecutive histidines for immuno-detection and affinity purification on nickel-agarose. Vector construction and analysis of transfected cells is described below.

The expression vectors described below were originally constructed from a clone designated pGEX-amR17-G produced during construction of pGEX- amR17 (described above) that included a previously undetected nucleotide deletion 5^' to the hRad 17 encoding sequence. (It is noted that the pGEX-amR17 expression vector described in Example 3 did include this nucleotide deletion.) The deletion was the loss of a guanosine nucleotide 3 ^' of the BamYQ. site which resulted in the loss of an Ncol site that was to have been created by the RAD 17.5 MOD PCR primer. The loss of this nucleotide meant that the hRadl7 sequence was originally cloned in a different reading frame with respect to upstream epitopes in expression vectors encoding the GFP-hRad 17 and FH-hRad 17 fusion proteins, pIΝD-GFP-amRl 7 and pIΝD-FH- amR17 (described below). The reading frame was corrected in each of these vectors by filling in the BamW site and the new clones were named pEΝD-GFP-R17ajnf and pIND-FH-Rl 7amf. The lower case nomenclature (amf) indicates that the hRad 17 insert was assembled from cDNA clones 586844 and 701704 and was modified at the 5' end for cloning purposes, and was filled in at the Bamrll site. The only difference between the R17amf insert and the amR17 insert (Example 3) was that there was one additional codon and two codon changes upstream of the hRad 17 cDNA, resulting in a slightly different linker region between hRad 17 and the amino terminal half of the fusion protein The polynucleotides and amino acid sequences for R17amf are set out in SEQ ID NOs 29 and 30, respectively.

The expression plasmid pIND-GFP-amR17 was constructed as follows The parental vector, pIND-βG-GLGFP, contained the β globin intron (to increase translation efficiency) inserted between the Λ7?el and Hwdlll restriction sites of vector pEND (Invitrogen), followed by the Green Lantern Green Fluorescent Protein (GLGFP) gene inserted between the Hwdlll and BamYU sites of pIND Vector pIND-βG-GLGFP was digested with BamrH and Xhol, gel-purified, and ligated to the Bam HJXhol gel-purified hRad 17 fragment from pGEX-amRl 7-G (described above)

The ligated DNA was transformed into E.coh TOPI OF^' cells (Invitrogen) and selected transformants were analyzed by restriction digestion (Ba H alone, Xhol alone, and BamWXhol together) It was during sequence analysis of the 5^' junction that first revealed the loss of a guanosine nucleotide preceding the hRad 17 gene (mentioned above) which resulted in the hRad 17 sequence being out of reading frame with the

GFP gene.

To correct the reading frame, pEND-GFP-amR17 was cleaved with BamHl and sticky ends were filled by T4 DNA polymerase in presence of dNTPs The blunt-ended plasmid was re-ligated with T4 DNA ligase and the sequence of the resulting clone, designated pEND-GFP-R17amf, was verified by DNA sequencing

Construction of pIND-FΗ-amR17 was performed as follows. A FLAG-6His (FH) cassette, encoding the FLAG epitope tag (Asp-Tyr-Lys-Asp-Asp- Asp-Asp-Lys, SEQ ED NO: 33) and six histidines cassette was originally cloned into vector pBAR an arabinose-inducible E.coh vector, to produce pBAR8A, from which a 128 bp Xbal/BamHl fragment encoding the FLAG tag and histidine encoding residues was isolated and gel purified. A ligation reaction was carried out with the following the gel purified DNA fragments: the 128 bp Xbal/BamHl FLAG-6His cassette, the BamHUXhol hRad 17-encoding sequence from pGEX-amR17-G, and pIND digested with Nhel and Xhol The ligation mix was transformed into E.coli TOPI OF' cells (Invitrogen) and plasmid preparations from selected transformants were analyzed by restriction digestion and DNA sequencing. The resulting plasmid was designated pIND-FH-amR17 Sequence of the 5^' junction also revealed the loss of the guanosine nucleotide preceding the hRad 17 sequence (discussed above) which resulted in the hRad 17 encoding sequence being out of reading frame with the upstream FH gene cassette To correct the reading frame, pIND-FH-amR17 was cleaved with BamHl and sticky ends were filled by T4 DNA polymerase in presence of dNTPs The blunt- ended plasmid was religated with T4 DNA ligase and the new clone pIND-FH-R17amf was verified by DNA sequencing

Protein Expression Stable pIND-GFP-Rl 7amf and pIND-FH-Rl 7amf transfectants in

ECR293 cells were generated ECR293 cells (Invitrogen) are a stably transfected cell line containing pVgRXR which encodes the ecdysone receptor In the presence of muristerone, the ecdysone receptor will activate transcription from the promoter on pIND ECR293 cells were grown in DMEM plus 10% fetal bovine serum (FBS) and penicillin, streptomycin, and 400 μg/ml zeocin to insure the presence of pVgRXR in the genome. Cells were transfected with 5 μg plasmid DNA (pIND-GFP-R17amf or pIND-FH-Rl 7amf) with Superfect Reagent (QIAGEN) Stable transfectants were selected in the presence of 400 μg/ml G418 after a 1 : 10 split onto 100 mm plates. Media was replaced every two to three days After 18 days of growth in G418 to select for transformed cells, 20 growth foci were isolated and transferred to 96 well dishes. After expansion of clones, cells were analyzed for induction of FH-Radl7 and GFP-Radl7 proteins by placing cells in media containing 1 μM muristerone. At twenty-one hours post-induction, cells were monitored under UV light for GFP fluorescence. GFP-R17amf fluorescence was observed in induced cells and its cellular localization was both the nuclear and cytoplasmic At 24 hours post-induction, cells from transfection plates (induced and uninduced) were harvested Cell extracts were prepared by resuspending ECR293 transfected cells in 0.2 ml Hypotonic Lysis Buffer (20 mM Tris-acetate, pH 8, 1.5 mM MgCl₂, 5 mM KCl, 1 mM DTT, 0.5 mM AEBSF, 2 μg/ml leupeptin, and 2 μg/ml aprotinin) and incubating on ice for 15 minutes, followed by sonication for 30 seconds. NaCl and EDTA were added to final concentrations of 250 mM and 2 mM, respectively. After a 30 minute incubation on ice, particulates were removed with a five minute centrifugation in an Eppendorf icrofuge Protein concentration of the cell extracts was determined by Coomassie Plus reagent (Pierce) with BSA as the standard

Western blot analysis (35 μg protein/lane) was used to detect the GFP- Rad 17 and FH-Rad 17 proteins Rad 17 rabbit antisera (see Example 4) detected both

100 kDa GFP-R17amf GFP-R17amf was also detected with GFP antisera (Clontech) In the absence of induction GFP-R17amf, was not detectable on Western blots FH- R17amf was not distinguishable from the 75 kD endogenous protein detected by the Rad 17 antisera Detection of FH-R17amf with a FLAG monoclonal antibody (Eastman Kodak) and a monoclonal antibody immunospecific for the histidine epitope

(Clontech) was inconclusive

Example 7 Analysis of hRad 17 ATPase Activity Recombinant hRad 17 was used in ATPase assays to assess the ability of the protein to hydrolyze ATP as predicted from the peptide sequence (see Example 1) despite the fact that there have been no published reports of ATPase activity attributed to yeast Rad 17 homologs However, subunits of replication factor C (RF- C, also called Activator 1). which are highly homologous to the Rad 17 protein family, have been shown to exhibit a DNA-dependent ATPase activity that is stimulated by the replication protein PCNA [Tsurimoto and Stillman, Proc. Natl Acad. Sci. (USA) 57.1023-1027 (1990)] Taking this into account, hRadl7 was monitored for ATPase activity in the presence or absence of DNA

ATPase assays were performed in buffer containing 25 mM Tris- acetate (pH 8), 10 mM magnesium acetate, 150 mM potassium acetate, and 5 mM β- mercaptoethanol, in the presence of ³²P-labeled ATP (0.06 μCi/μl) at concentrations ranging from 10 to 250 μM GST-amRl 7 was tested at 1 4 to 26 ng/μl concentrations Reactions were incubated at 37° C for set times ranging from 15 to 45 minutes Reactions using [γ-³²P]ATP were stopped with an equal volume of 20 mM EDTA Reactions using [α-³²P]ATP were stopped with 0 7 volumes of 24 mM ATP,

ADP, AMP and EDTA An aliquot of each reaction (1 μl) was spotted on a PEI cellulose F TLC plate (EM Separations) which was developed in 1 M LiCl and dried before autoradiography Quantitation was performed by excising the unhydrolyzed ATP and the hydrolysis product from the TLC plate, placing each separately in 5 ml scintillation fluid and determining counts per minute (cpm) in a scintillation counter In experiments with [γ-³²P]ATP, TLC plates were aligned with autoradiograms to determine the position of the ATP and released phosphate With [α-³ P]ATP experiments, the location of the ATP and ADP was visualized on the TLC plates under 254 nm light due to the presence of these nucleotides in the stop mix

Results revealed that GST-amRl 7 had ATPase activity as monitored by the release of labeled phosphate from [γ-³²P]ATP and of labeled ADP from [α-

³²P]ATP The conversion of ATP to ADP and phosphate is the standard hydrolysis reaction for ATPases with Walker A box motifs No further hydrolysis of ADP was detected The reaction rate of GST-amRl 7 ranged from 3 to 13 nM/sec in reactions with 26 ng/μl protein. Because GST-amRl 7 was not a homogeneous protein preparation (see Example 3) and the ATPase activity of the breakdown products could not be predicted, the true rate constant (moles ATP hydrolyzed per mole GST-amRl 7 per second) could not be precisely calculated In GST-amRl 7 preparation with proportionately more full length protein (approximately 105 kDa), proportionately more ATPase activity was observed Several variables in the reaction conditions were tested The presence of DNA was tested by adding either 250 nM of a NotVXhol linker, 250 nM oligonucleotide dT₍₁₂₎, 12 μM (total nucleotide) of a single stranded DNA polymer (poly-dA) or both poly-dA and oligonucleotide dT₍₁₂₎ Stimulation of the GST-amR17 ATPase by DNA was not observed in any of these reactions. In reactions with 25 μM ATP, an assortment of unlabeled nucleotides (at 1 mM) were added to monitor their abilities to act as competitors Under these conditions, ADP was found to be an effective inhibitor (83%) of the GST-amRl 7 ATPase, while dATP and GTP (36% inhibition), and AMP and adenosine (20% inhibition) were less effective Electrolyte concentrations ranging from 0 to 200 mM potassium acetate did not substantially affect the activity of GST-amR 17

The protein preparation of the Walker A box point mutant, GST- amRl 7.KE, did have ATPase activity Whether this activity was innate to GST- amR17.KE or a bacterial contaminant has not been yet determined. However, the ATPase activity of GST-amRl 7. KE differs from that in GST-amRl 7 in two significant ways. First, unlike GST-amRl 7, the GST-amRl 7. KE activity is not inhibited in the presence of excess ADP. Second, the reaction rate of GST-amRl 7 remained relatively constant in the range of 10 to 100 μM ATP, while the GST-amRl 7. KE reaction rate increased with increasing ATP concentration. This latter result indicates that GST- amRl 7 has a much lower Km for binding ATP than does GST-amRl 7.KE (or the contaminating activity in this preparation).

Photo-crosslinking of an ATP analog (8-azido-[i-³²P]ATP) to GST- amR17 and GST-amRl 7. KE is used as a separate assay to assess the ATP-binding properties of these proteins. The protocol follows published guidelines [Jakob, et al, J. Biol Chem. 271: 10035-10041 (1996)].

Example 8 Identification of hRadl7 Binding Partners

The yeast two-hybrid system was used to identify protein-protein interactions between hRad 17 and a some specific human checkpoint proteins ( ATR,

Chkl, and hRad 17) as well as mammalian proteins encoded in a cDNA library. The yeast two-hybrid system has previously been described [Hollenberg, et al, Moil. Celliol 75:3812-3822 (1995)]. The yeast strain L40, which contains multiple LexA binding sites upstream of the HIS3 and β-galactosidase genes, was transformed with two gene-hybrid vectors that initiate the expression of fusion proteins containing the LexA DNA-binding domain (pBTMl 16) and the GAL4 transcriptional activation domain (pACT2). Interaction between the LexA fusion protein and the GAL4 fusion protein is scored by expression of HIS3 (growth without histidine) and of β- galactosidase (blue color in the presence of X-GAL). Additionally, 3-amino-triazole can be added to the growth media to make selection for EHS3 expression more stringent.

The EcoRI/λ7?oI hRad 17 fragment from pGΕX-amR17 was ligated between the EcoRI and Xhol sites of pBTM 1 16 to create pBTM 1 16-Rad 17. An

EcoKUSall Chkl gene fragment [Flagg, et al, Curr.Biol, 7:977-986 (1997) was ligated into the EcoKUSall sites of pBTMl 16 to create pBTMl 16-Chkl. Additionally, 4 different gene fragments from ATR have been cloned into pBTM l 16 to create pBTMl 16-ATR AB, -ATR/CD, -ATR/EF. and -ATR GH A similar strategy was employed to create pACT2-Radl 7 and pACT2-Chkl (genes were cloned between EcoKUXhol sites of pACT2), except that an ΕcoRI linker (5'- AATTCGGCGCGCCG-3') was used to place the Chkl sequence in the correct reading frame and the hRad 17 gene fragment came from pGΕX-amR17-G (see Example 6). Correct reading frame of the constructs was verified by DNA sequencing Expression of the fusion proteins in L40 yeast was verified on Western blots using LexA-specific and GAL4-specific antibodies. Co-transformation of different plasmid pairs into L40 has indicated that hRad 17 does not bind to Chkl. the four different ATR domains, or to itself

Transformation of L40 containing pBTMl 16-Radl7 with cDNA fusion libraries (human testis MatchMaker library, Clontech) and a human embryo cDNA library with the VP16 transcriptional activation domain was performed to identify hRad 17-interacting proteins.

Between 30 and 50 positive interactors were isolated from each library, and are undergoing further analysis (DNA sequencing and specificity tests using the two hybrid system).

Claims

What is claimed is

1 A purified and isolated human rad 17 polypeptide

2 The polypeptide according to claim 1 compπsing the rad 17 amino acid sequence set out in SEQ ID NO 2

3 A polynucleotide encoding the polypeptide according to claim 1 or 2

4 The polynucleotide according to claim 3 compπsing the sequence set forth in SEQ ED NO 1

5 A polynucleotide encoding a human rad 17 polypeptide selected from the group consisting of a) the polynucleotide according to claim 2, b) a DNA which hybridizes under highly stringent conditions to the complement of the polynucleotide of (a), and c) a DNA which would hybridize to the polynucleotide of (a) but for degeneracy of the genetic code

6 The polynucleotide of claim 5 which is a DNA molecule

7 The DNA of claim 6 which is a cDNA molecule

8 The DNA of claim 6 which is a genomic DNA molecule

9 The DNA of claim 6 which is a wholly or partially chemically synthesized DNA molecule

10 An anti-sense polynucleotide which specifically hybridizes with the complement of the polynucleotide of claim 5

1 1 A expression construct comprising the polynucleotide according to claim 5

12 A host cell transformed or transfected with the polynucleotide according to claim 1 1

13 A method for producing a human rad 17 polypeptide comprising the steps of a) growing the host cell according to claim 12 under conditions appropriate for expression of the human radl 7 polypeptide and b) isolating the rad 17 polypeptide from the host cell or the medium of its growth

14 An antibody specifically immunoreactive with the polypeptide according to claim 1 or 2

15 The antibody according to claim 14 which is a monoclonal antibody

16 A hybridoma which secretes the antibody according to claim 15

17 An anti-idiotype antibody specifically immunoreactive with the antibody according to claim 14

18 A method to identify a specific binding partner compound of the human rad 17 polypeptide according to claim 1 or 2 comprising the steps of a) contacting the rad 17 polypeptide with a compound under conditions which permit binding between the compound and the rad 17 polypeptide, b) detecting binding of the compound to the rad 17 polypeptide, and c) identifying the compound as a specific binding partner of the rad 17 polypeptide

19 The method according to claim 18 wherein the specific binding partner modulates activity of the rad 17 polypeptide

20 The method according to claim 19 wherein the compound inhibits activity of the rad 17 polypeptide

21 The method according to claim 19 wherein the compound enhances activity of the rad 17 polypeptide

22 A compound identified by the method according to claim 18

23 A method to identify a specific binding partner compound of the rad 17 polynucleotide according to claim 5 comprising the steps of. a) contacting the hRad 17 polynucleotide with a compound under conditions which permit binding between the compound and the rad 17 polynucleotide; b) detecting binding of the compound to the rad 17 polynucleotide, and c) identifying the compound as a specific binding partner of the rad 17 polynucleotide

24 The method according to claim 23 wherein the specific binding partner modulates expression of a rad 17 polypeptide encoded by the hRad 17 polynucleotide

25 The method according to claim 24 wherein the compound inhibits expression of the rad 17 polypeptide

26 The method according to claim 24 wherein the compound enhances expression of the rad 17 polypeptide

27 A compound identified by the method according to claim 23

28 A composition comprising the compound according to claim 27 and a pharmaceutically acceptable carrier

29 A method to identify modulators of rad 17 enzymatic activity comprising the steps of a) contacting a rad 17 and a substrate in the presence and absence of a compound, b) measuring rad 17 activity on the substrate in the presence and absence of the compound, and c) identifying the compound as a modulator of rad 17 activity by detecting a change in rad 17 activity on the substrate in the presence of the compound

30 The method according to claim 29 wherein the rad 17 activity is ATPase activity

31 The method according to claim 30 where the compound is identified as an inhibitor of rad 17 enzymatic activity on the substrate when decreased rad 17 activity is detected in the presence of the compound

32. The method according to claim 30 where the compound is identified as an activator of rad 17 enzymatic activity on the substrate when increased rad 17 activity is detected in the presence of the compound

33. A compound identified by the method according to any one of claims 29, 30, 31, and 32.

34. A composition comprising the compound according to claim 33 and a pharmaceutically acceptable carrier.