WO1999011795A1 - Mammalian chk1 effector cell-cycle checkpoint protein kinase materials and methods - Google Patents

Abstract

The present invention generally relates to genes encoding cell cycle checkpoint kinase related proteins essential to meiosis, mitosis, and DNA damage responses in cells and the respective proteins. These kinases arrest the cell cycle following DNA damage to allow DNA repair prior to mitosis, meiosis, or initiation of DNA replication. More particularly, the invention provides a novel cycle checkpoint kinase, Chkl, and polynucleotide sequences encoding Chkl. Assays for identifying modulators of Chkl are also disclosed. Modulators are useful for example, in chemotherapy and as radiation adjuvants.

Description

MAMMALIAN CHK1 EFFECTOR CELL-CYCLE CHECKPOINT PROTEIN KINASE MATERIALS AND METHODS

FIELD OF THE INVENTION

The present invention generally cell cycle checkpoint protein kinases which are essential to cellular DNA damage responses and coordinating cell cycle arrest. The checkpoint kinases play a role in the surveillance and response to DNA damage that occurs as a result of replication errors, DNA mismatches, radiation treatment, or chemotherapy. These checkpoint kinases are required in regulatory pathways that lead to cell cycle arrest and apoptosis following DNA damage, giving the cell notice and time to correct lesions prior to the initiation of DNA replication or chromosome separation. More particularly, the present invention relates to novel mammalian effector (Chkl) checkpoint protein kinases, polynucleotides encoding the same, and methods and materials for assaying and modulating the enzymatic activity of the kinases.

BACKGROUND

The cell cycle is structurally and functionally conserved in its basic process and mode of regulation across all eukaryotic species. The process of eukaryotic cell growth and division is the somatic (mitotic) cell cycle which consists of four phases, the

Gl phase, the S phase, the G2 phase, and the M phase. The Gl, S, and G2 phases are collectively referred to as interphase of the cell cycle. During the Gl (gap) phase, biosynthetic activities of the cell progress 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 itself splits into two daughter cells each receiving one nucleus containing one of the two sets of chromosomes. Mitosis (the M phase of the cell cycle) is immediately followed by cytokinesis.

Cytokinesis terminates the M phase and marks the beginning of interphase of the next cell cycle. The sequence in which the events in the cell cycle proceed is tightly regulated such that the initiation of one cell cycle event is dependent on the completion of the prior cell cycle event. This allows fidelity in the duplication and segregation of genetic material from one generation of somatic cells to the next.

Meiosis is the form of cell division that produces germ cells in higher eukaryotes. In contrast to mitosis, where mitotic cell division results in genetically identical cells containing two of each chromosome, meiotic cell division results in cells containing one copy of each chromosome. In addition, in meiosis homologous chromosomes pair and exchange genetic material. Meiosis consists of two stages of cell division, meiosis I and meiosis II. In meiosis I, maternal and paternal chromosomes duplicate and homologous chromosomes pair together (synapsis). The cell then undergoes division in which homologous pairs of duplicate chromosomes separate and enter individual cells resulting in two diploid daughter cells. The daughter cells then enter meiosis II. In meiosis II, the chromosomes align without further replication and sister chromatids separate, as in mitosis, to produce haploid cells. The sequence of events in both meiosis I and meiosis II are interphase, prophase, metaphase, anaphase, and telophase.

The first stage of meiosis I is interphase I in which each chromosome is replicated. The two copies of the replicated chromosome are called sister chromatids. Five sequential stages then define the first meiotic prophase. During leptotene, the newly replicated sister chromatids are in close apposition so that they may associate and undergo recombination. During zygotene, a proteinaceous structure termed the synaptonemal complex forms between maternal sister chromatids and paternal sister chromatids resulting in a bivalent (four chromatids). During pachytene, recombination between two sister chromatids (i.e. exchange of genetic material between maternal and paternal chromosomes) begins. The next stage, diplotene, is marked by the disassembly of the protein axes and the two sister chromatids begin separating. Diakinesis, the final stage, is characterized by detachment of the chromosomes from the nuclear envelope and each bivalent is clearly seen to contain four separate chromatids, with each pair of sister chromatids linked at their centromeres. Thus, early in meiosis during the "reduction division" process, sister chromatids pair and undergo reciprocal recombination at some regions. Programmed DNA strand breaks initiate recombination. [Cao et al., Cell, 88:375-384 (1997)]. The changes observed during the first meiotic prophase facilitates the genetic reassortment that assures genetic viability.

The process of monitoring genome integrity and preventing cell cycle progress in the event of DNA damage has been described as "cell cycle checkpoint" [Hartwell and Weinert Science, 246:629-634 (1989); Weinert et al., Genes and Dev.,

8:652 (1994)]. Cell cycle checkpoints consist of signal transduction cascades which couple DNA damage detection to cell cycle progression. In meiosis, cell cycle checkpoints control programmed DNA breaks, ensuring the proper segregation of a complete haploid set of chromosomes to each gamete. Failure of cell cycle checkpoints predisposes individuals to or directly causes many disease states such as cancer, ataxia telangiectasia, 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.

It has been reported that cell cycle checkpoints comprise at least three distinct classes of polypeptides which act sequentially in response to cell cycle signals or defects in chromosomal mechanisms. [Carr, A.M., Science, 271:314-315 (1996)]. The first class is a family of proteins which detect or sense DNA damage or abnormalities in the cell cycle. These sensors include Atm and Atr [Keegan et al., Genes and Devel.,

10:2423-2437 (1996)]. The second class of polypeptides amplify and transmit the signal detected by the detector and is exemplified by Rad53 [Alen et al. (1994) Genes Dev. 8:2416-2488]. Finally, the cell cycle checkpoint effects a cellular response, e.g. arrest of mitosis/meiosis, apoptosis through cell cycle effectors. Genetic analysis in the yeasts Schizosaccharomyces pombe and

Saccharomyces cerevisiae has identified a number of checkpoint genes important for mitotic arrest and DNA repair responses to IR. For a review, see Carr and Hoekstra, Trends in Cell Biology, 5: 32-40 (1995). One such gene, identified in yeasts, is required for a DNA damage checkpoint which arrests mitosis 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 is named rad3 in S. pombe [Seaton et al., Gene, 119: 83-89 (1992) Bentley et al., (1996) EM BO J. 15: 6641-6651], MEC1 ESR1 in S. cerevisiae [Kato and Ogawa, Nuc. Acids. Res., 22(15): 3104-3112 (1994)], but is hereinafter referred to as rad3. Cells having mutations in rad3 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., IR, DNA damaging agents, and mutations affecting chromosomal integrity). See Weinert et al., GENES & DEVELOPMENT, 8: 652-665 (1994) and Al-Khodairy et al., EMBO J., 11(4): 1343-1350 (1992). Rad3 thus appears to be a checkpoint detector of DNA damage (Carr, 1996). In addition rad3 appears to function in vivo as a multimer. [Bentley et al., 1996]. The product of the rad3 gene is an approximately 270 kD protein that is a member of a growing family of high molecular weight mammalian checkpoint kinases. See Hunter, Cell, 83: 1-4 (1995) for a discussion of this family of kinases. This family includes ecll (S. cerevisiae) mei-41 (Drosophila melanogaster), tori (S. cerevisiae), tor2 (S. cerevisiae), Frap (Human), tell (S. cerevisiae), DNA-Pk (Human) Atr (human) and Atm (human). These proteins have been identified as members of a family based on sequence homology and complementation studies.

The human homolog of rad3, Atr (Ataxia Telangiectasia and rad3 related) was identified in Bently et al., EMBO J., 15:6641-6651 (1996). Bently et al., showed that recombinant Atr can heteromultimerize with rad3 when expressed in S. pombe. In addition, recombinant Atr expression complemented S. cerevisiae mecl mutants.

The primary structures of the catalytic domains found in members of this kinase family are closely related to well characterized phosphatidylinositol kinases. This structural relationship initially suggested that these mammalian checkpoint kinases might be capable of phosphorylating lipids. However, when the substrate specificity of the mammalian checkpoint kinases is examined, these enzymes appear to function as protein kinases and have yet to be demonstrated to phosphorylate phosphatidylinositides.

Atm (Ataxia Telangiectasia Mutated), another member of this family was identified through the analysis of the human disease syndrome ataxia-telangiectasia (AT) [Savitsky et al., Science, 268: 1749-1753 (1995) and Savitsky et al., Human Molecular Genetics, 4(11):2025-2032 (1995)]. Patients with AT exhibit a diverse set of clinical symptoms, including predisposition to a variety of tumor types. Fibroblasts from AT patients are radiosensitive and fail to undergo mitotic arrest following treatment with IR. Mutant mice lacking Atm show gonadal atrophies, meiotic abnormalities and severe chromosome fragmentation [Ashley et al., Proc. Nat. Acad. Sci. USA, 93:13084 (1996)]. This is reminiscent of the S. pombe stains with rad3 defects where cells fail to sense or respond appropriately to DNA damage.

This family of kinases thus appear to function as detectors for defects of various cell-cycle transitions [Carr, 1996].

Recently it was shown that the mammalian Atm and Atr proteins associate with chromosomes during pachynema of meiotic prophase and may monitor strand disruptions that occur during meiotic chromosome synapsis and recombination.

Localization of Atm and Atr kinases shows complementary patterns of foci during zygonema and pachynema, commensurate with different roles in monitoring the DNA structure during meiotic recombination [Keegan et al., Genes Dev., 10:2423 (1996)].

Checkpoint proteins clearly play a role in signal transduction cascades. The detectors Atm and Atr are protein kinases that comprise a early step in the signal transduction cascade. It is thought that signally is amplified by protein kinases such as Rad53 which acts to transduce a signal from the detectors to the effectors.

To date, p53 and S. pombe Chkl and weel have been identified as effector checkpoint proteins. The S. pombe Chkl gene was isolated based on its genetic interaction with the cdc2.r4 allele [Al-Khodairy et al., Mol. Biol. Cell, 5:147-160 (1994)], and by complementation of the radiation sensitivity of a rad27 mutant. cdc2 encodes a protein kinase subunit that associates with cyclins to form active protein kinase complexes that induce passage through mitosis. [Broek et al., Nature, 349:388-393 (1991]. S. pombe Chkl appears to effect the mitotic arrest following DNA damage and S. pombe Chkl deletion mutants fail to undergo cell-cycle arrest after irradiation [Walworth et al. , Nature, 363:368 (1993), Al-Khodairy et al., Mol. Biol. Cell., 5:147-160 (1994), Carr, A.M., Semin. Cell Biol., 6:65-72 (1995)]. However, Chkl is not required for the other checkpoint protein-mediated DNA cellular responses to blocks to DNA replication. Walworth and Bernards Science, 271 :353-356 (1996) demonstrated that in vivo activity of Chkl is regulated by phosphorylation of Chkl. By examining the phosphorylation status of Chkl in various S. pombe strains harboring mutations in checkpoint genes, Walworth and Bernards, Science, 271 :353-356 (1996) demonstrated that Chkl acts downstream of these checkpoint proteins. Chkl phosphorylation was abolished or greatly diminished in S. pombe rad 1, rad 3, rad 9, rad 17, and rad26 mutants. In addition, S. pombe Chkl appears to function during the Gl and G2 phases of mitosis. Carr et al., Curr. Biol., 5:1179-1190 (1995) demonstrated that Chkl deficient cells failed to enter the S phase indicating that Chkl is a Gl checkpoint kinase. It was demonstrated in O'Connell et al., EMBO Journal, 16:545-554 (1997) that Chkl phosphorylated weel, a checkpoint kinase involved in G2 cell cycle arrest. The Drosophila homolog of S. pombe Chkl was identified as "Grp" in

Sibon et al., Nature, 388:93-97 (1997). Grp is required for cell cycle control at the mid-blastula transition (MBT) in which the maternal component of the DNA-replication machinery slows DNA synthesis and induces a checkpoint-dependent delay in cell cycle progression during embryogenesis. The C. elegans homolog of Chkl was first reported as an EST in Genbank

Accession No. U44902. The S. cerevisiae homolog of Chkl was identified as a probable ser/thr protein kinase in Genbank Accession No. 585344.

To date, there has been no identification of a mammalian effector checkpoint (Chkl) protein kinase. There thus exists a need in the art for identification of the mammalian effector 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 isolation of polynucleotides encoding those proteins which in themselves may be useful as therapeutics or which would enable the development of therapeutically useful modulators of the proteins encoded by the polynucleotides.

SUMMARY OF THE INVENTION The present invention provides novel human mammalian effector cell cycle checkpoint, Chkl, kinases and polynucleotides encoding the same.

In one of its aspects, the present invention provides purified and isolated polynucleotides (e.g., DNAs and RNAs, both coding and non-coding strands thereof) encoding the human and mouse effector cell cycle checkpoint kinase and polynucleotides encoding other mammalian checkpoint kinases that exhibit 50% or greater amino acid identity to the polynucleotide region encoding the human Chkl kinase domain (amino acids 14 to 264 of SEQ ID NO.: 2). Preferably, the polynucleotides encode a checkpoint kinase that exhibits 70% or greater amino acid identity to amino acids 14 to 264 of SEQ ID NO.: 2. Even more preferably, the polynucleotides encode a checkpoint kinase that exhibits 90% or greater amino acid identity to amino acids 14 to 264 of SEQ ID NO.: 2. Polynucleotides contemplated by the invention include genomic DNAs, RNAs, cDNAs and wholly or partially chemically synthesized DNAs. Preferred polynucleotides of the invention comprise the human Chkl DNA sequence set out in SEQ LD NO.: 1, the mouse Chkl DNA sequence set out in SEQ LD NO. : 3, and DNA sequences which hybridize to the noncoding strands thereof under stringent conditions or which would hybridize but for the redundancy of the genetic code. Exemplary stringent hybridization conditions are as follows: hybridization at 65° C in 3X SSC, 20 mM NaPO₄ pH 6.8 and washing at 65° C in 0.2X SSC. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). A polynucleotide vector encoding human Chkl (plasmid pGEMT-Chklhu ) was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 on date August 27, 1997 under Accession No. ATCC98520.

The DNA sequence information provided by the present invention makes possible the identification and isolation of DNAs encoding mammalian checkpoint related molecules by well-known techniques such as DNA/DNA hybridization as described above and polymerase chain reaction (PCR) cloning. As one series of examples, knowledge of the sequence of a cDNA encoding a mammalian Chkl of the invention makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences encoding the kinase and expression control regulatory sequences such as promoters, operators and the like. Similarly, knowledge of a partial cDNA sequence makes isolation of a complete cDNA possible. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of the kinase non-human species enzymes homologous to the mammalian Chkl kinase and other structurally related proteins sharing one or more of the enzymatic activities, or abilities to interact with members or regulators, of the cell cycle checkpoint pathway in which mammalian Chkl participates. Polynucleotides of the invention when detectably labeled are also useful in hybridization assays to detect the capacity of mammalian cells to synthesize kinases of the invention. The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout" strategies [see, Capecchi, Science, 244: 1288-1292 (1989)], of rodents that fail to express a functional kinase or that express a variant thereof. Such rodents and their cells are useful as models for studying the activities of mouse and kinase modulators in vivo. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the mammalian Chkl gene locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of Chkl by those cells which ordinarily express the same.

For example, primers designed from the Chkl cDNA are useful for reverse transcriptase PCR analysis of mRNA samples from tumor cells to detect the presence or absence of Chkl mRNA. Further, sequence information from the Chkl genomic clone can be used for single stranded conformational polymorphism (SSCP) analysis of genomic DNA prepared from tumor cells to detect alterations or mutations of the Chkl gene.

Likewise, the Chkl cDNA and/or the Chkl genomic clone can be used in fluorescence in situ hybridization (FISH) analysis to detect alterations in the Chkl gene.

The invention also provides autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating polynucleotides of the invention, especially vectors in which the polynucleotides are functionally linked to an endogenous or heterologous expression control DNA sequence and a transcription terminator.

According to another aspect of the invention, host cells, especially unicellular host cells such as procaryotic and eukaryotic cells, are stably transformed or transfected with DNAs of the invention in a manner allowing expression of a mammalian

Chkl kinase therein. Host cells of the invention are conspicuously useful in methods for the large scale production of Chkl wherein the cells are grown in a suitable culture medium and the desired enzymes are isolated from the cells or from the medium in which the cells are grown.

Chkl products having part or all of the amino acid sequence set out in SEQ ID NO: 2 or SEQ ID NO.: 4 are contemplated. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., myristoylation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. The enzyme products of the invention may be full length polypeptides, fragments or variants. Variants comprise Chkl products wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of the protein kinase activity specific to Chkl; or (2) with disablement of the protein kinase activity specific to Chkl; or (3) with disablement of the ability to interact with members or regulators of the cell cycle checkpoint pathway. Substrates of Chkl and proteins which interact with Chkl may be identified by various assays.

Substrates of Chkl may be identified by incorporating test compounds in assays for kinase activity. Chkl kinase is resuspended in kinase buffer and incubated either in the presence or absence of the test compound (e.g., myelin basic protein, casein, histone HI, or appropriate substrate peptide). The amount of phosphate transferred by the kinase to the test compound are measured by autoradiography or scintillation counting. Transfer of phosphate to the test compound is indicative that the test compound is a substrate of the kinase.

Yet another aspect of this invention provides a diagnostic assay for detecting and quantifying the presence of Chkl in a biological sample. A biological sample suspected of comprising Chkl is utilized in a kinase assay. As described herein, the presence of Chkl is identified by the phosphorylation of a substrate protein, e.g. myelin b protein, or the detection of a self-phosphorylated product. The phosphorylated product of the kinase reaction can be detected by for example, autoradiography or scintillation counting. Interacting proteins may be identified by the following assays.

A first assay contemplated by the invention is a two-hybrid screen. The two-hybrid system was developed in yeast [Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)] and is based on functional in vivo reconstitution of a transcription factor which activates a reporter gene. Specifically, a polynucleotide encoding a protein that interacts with Chkl is isolated by: transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain; expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of Chkl and either the DNA binding domain or the activating domain of the transcript ion factor; expressing in the host cells a library of second hybrid DNA sequences encoding second fusions of part or all of putative Chkl binding proteins and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion; detecting binding of an Chkl interacting protein to Chkl in a particular host cell by detecting the production of reporter gene product in the host cell; and isolating second hybrid DNA sequences encoding the interacting protein from the particular host cell. Presently preferred for use in the assay are a lexA promoter to drive expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the lexA DNA binding domain and the GAL4 transactivation domain, and yeast host cells. Other assays for identifying proteins that interact with Chkl may involve immobilizing Chkl or a test protein, detectably labeling the nonimmobilized binding partner, incubating the binding partners together and determining the amount of label bound. Bound label indicates that the test protein interacts with Chkl.

Another type of assay for identifying Chkl interacting proteins involves immobilizing Chkl or a fragment thereof on a solid support coated (or impregnated with) a fluorescent agent, labeling a test protein with a compound capable of exciting the fluorescent agent, contacting the immobilized Chkl with the labeled test protein, detecting light emission by the fluorescent agent, and identifying interacting proteins as test proteins which result in the emission of light by the fluorescent agent. Alternatively, the putative interacting protein may be immobilized and Chkl may be labeled in the assay. Also comprehended by the present invention are antibody products (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and antigen-binding fragments thereof) and other binding proteins (such as those identified in the assays above) which are specific for the Chkl kinases of the invention. Binding proteins can be developed using isolated natural or recombinant enzymes. The binding proteins are useful, in turn, for purifying recombinant and naturally occurring enzymes and identifying cells producing such enzymes. Assays for the detection and quantification of proteins in cells and in fluids may involve a single antibody substance or multiple antibody substances in a "sandwich" assay format to determine cytological analysis of Chkl protein levels. The binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting, or stimulating) enzyme/substrate or enzyme/regulator interactions. Anti-idiotypic antibodies specific for mammalian checkpoint kinase binding proteins are also contemplated.

It is further contemplated that antibodies against Chkl can be used in diagnosis of Atm function. Because Chkl protein levels are low or absent in AT patients

Chkl levels can be used as a marker in the development of compounds that inhibit Atm function. Because expression of Chkl in AT cells is low or non-existent, inhibition of Atm function should decrease Chkl expression. This decrease can be monitored by examining the expression levels of Chkl. The invention contemplates that mutations in the Chkl gene that result in loss of normal function of the Chkl gene product underlie human disease states in which failure of a cell cycle checkpoint is involved. Gene therapy to restore Chkl activity would thus be indicated in treating those disease states (for example, testicular cancer). Delivery of a functional Chkl gene to appropriate cells is effected in vivo or ex vivo by use of viral vectors (e.g., adenovirus, 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 Chkl 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 Chkl. Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to Chkl expression control sequences or Chkl 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 Chkl 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.

Checkpoint signal transduction results in transcriptional regulation. One example of transcriptional regulation is MyoD muscle regulation. Chkl expression suppresses the ability of MyoD to induce muscle gene transcription and suppresses the ability of MyoD to induce myogenesis (see Example 7). It is contemplated that another aspect of this invention is to regulate Chkl levels in order to effect the differentiation and proliferation of stem cells such as those involved in muscle proliferation. Agents that modulate Chkl protein kinase activity may be identified by incubating a test compound with Chkl immunopurified from cells naturally expressing the mammalian checkpoint protein kinase, with Chkl obtained from recombinant procaryotic or eukaryotic host cells expressing the enzyme, or with purified Chkl, and then determining the effect of the test compound on Chkl protein kinase activity. The activity of the checkpoint protein kinase can be measured by determining the amount of

32P-phosphate transferred by the protein kinase from gamma-32P-ATP to either itself (autophosphorylation) or to an exogenous substrate such as a lipid or protein. The amount of phosphate incorporated into the substrate is measured by scintillation counting or autoradiography. An increase in the amount of phosphate transferred to the substrate in the presence of the test compound compared to the amount of phosphate transferred to the substrate in the absence of the test compound indicates that the test compound is an activator of the Chkl protein kinase. Conversely, a decrease in the amount of phosphate transferred to the substrate in presence of the test compound compared to the moles of phosphate transferred to the substrate in the absence of the test compound indicates that the modulator is an inhibitor of the Chkl protein kinase. In a presently preferred assay, a Chkl -specific antibody linked to agarose beads is incubated with a cell lysate prepared from host cells expressing the protein kinase. The beads are washed to remove proteins binding nonspecifically to the beads and the beads are then resuspended in kinase buffer. The reaction is initiated by the addition of gamma-32P-ATP and an appropriate exogenous substrate such as lipid or peptide. The activity of the protein kinase is measured by determining the moles of 32P -phosphate transferred either to the protein kinase itself or the added substrate.

In a preferred embodiment the host cells lack endogenous Chkl and/or ATM protein kinase activity. The selectivity of a compound that modulates the protein kinase activity of Chkl can be evaluated by comparing its activity on Chkl to its activity on other known mammalian checkpoint protein kinases. The combination of the recombinant Chkl products of the invention with other recombinant mammalian checkpoint kinase products in a series of independent assays provides a system for developing selective modulators of Chkl. Furthermore, combinatorial libraries, peptide and peptide mimetics, defined chemical entities, oligonucleotides, and natural product libraries may be screened for activity as modulators in assays such as those described below.

For example, an assay for identifying modulators of Chkl kinase activity involves incubating a Chkl protein kinase preparation in kinase buffer with gamma-32P-ATP and an exogenous kinase substrate, both in the presence and absence of a test compound, and measuring the amount of phosphate transferred to the substrate. An increase in the amount of phosphate transferred to the substrate in presence of the test compound compared to the amount of phosphate transferred to the substrate in the absence of the test compound indicates that the test compound is an activator of the Chkl kinase. Conversely, a decrease in the amount of phosphate transferred to the substrate in presence of the test compound compared to the amount of phosphate transferred to the substrate in the absence of the test compound indicates that the modulator is an inhibitor of said Chkl protein kinase.

Moreover, assays for identifying compounds that modulate interaction of Chkl with other proteins may involve: transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain; expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of Chkl and the DNA binding domain or the activating domain of the transcription factor; expressing in the host cells a second hybrid DNA sequence encoding part or all of a protein that interacts with Chkl and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion; evaluating the effect of a test compound on the interaction between Chkl and the interacting protein by detecting binding of the interacting protein to Chkl in a particular host cell by measuring the production of reporter gene product in the host cell in the presence or absence of the test compound; and identifying modulating compounds as those test compounds altering production of the reported gene product in comparison to production of the reporter gene product in the absence of the modulating compound. Presently preferred for use in the assay are a lexA promoter to drive expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the lexA DNA binding domain and the GAL4 transactivation domain, and yeast host cells.

Another type of assay for identifying compounds that modulate the interaction between Chkl and an interacting protein involves immobilizing Chkl or a natural Chkl interacting protein, detectably labeling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the present of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of Chkl interaction with protein. Conversely, an increase in the binding in the presence of the test compound compared to the amount label bound in the absence of the compound indicates that the putative modulator is an activator of Chkl interaction with the protein.

Yet another method contemplated by the invention for identifying compounds that modulate the binding between Chkl and an interacting protein involves immobilizing Chkl or a fragment thereof on a solid support coated (or impregnated with) a fluorescent agent, labeling the interacting protein with a compound capable of exciting the fluorescent agent, contacting the immobilized Chkl with the labeled interacting protein in the presence and absence of a test compound, detecting light emission by the fluorescent agent, and identifying modulating compounds as those test compounds that affect the emission of light by the fluorescent agent in comparison to the emission of light by the fluorescent agent in the absence of the test compound. Alternatively, the Chkl interacting protein may be immobilized and Chkl may be labeled in the assay. Modulators of Chkl may affect its protein kinase activity, its localization in the cell, and/or its interaction with members of the cell cycle checkpoint pathway. Chkl modulators may be formulated in compositions comprising pharmaceutically acceptable carriers. Such compositions may additionally include chemotherapeutic agents. Dosage amounts indicated would be sufficient to result in modulation of Chkl activity in vivo. Selective modulators may include, for example, polypeptides or peptides which specifically bind to Chkl or Chkl nucleic acid, oligonucleotides which specifically bind to Chkl or Chkl nucleic acid, and/or other non-peptide compounds (e.g., isolated or synthetic organic molecules) which specifically react with Chkl or Chkl nucleic acid. Mutant forms of Chkl which affect the enzymatic activity or cellular localization of wild-type Chkl are also contemplated by the invention.

DETAILED DESCRIPTION

The present invention is illustrated by the following examples. Example 1 details the isolation of polynucleotides encoding mammalian Chkl kinases and chromosomal mapping of the human Chkl DNA. Example 2 describes the recombinant expression of DNAs encoding mammalian Chkl . Example 3 describes the preparation of antibodies to Chkl. Northern blots showing tissue and cell distribution of Chkl are described in Example 4. Example 5 reports the results of immunohistological and western blot studies of Chkl expression. Example 6 describes biochemical and biological activities of murine Chk 1.

Example 1 A. Isolation of Human Chkl cDNA

A ChklHu cDNA was identified by screening EST sequences for similarity to S. pombe Chkl . An EST (H67490) with homology to the COOH-terminus of Chkl was identified and cloned and used to build a contig showing limited homology to the COOH-terminal 120 amino acids. This contig was extended by RACE PCR to give a clone of 1735bp. The RACE PCR fragment was used to probe cDNA libraries to generate the final sequence. Library screening of 1 x 10⁶ independent cDNA clones from a human testis unizap library (Clontech) with RACE derived sequence information at high stringency in Express hybridization solution (Clontech) yielded eleven overlapping sequences that were used to assemble the full length ChklHu.

The full length human Chkl cDNA was subcloned into pGEMT. The plasmid containing the full length human cDNA is identified as pGEMT-ChklHU pGEMT-ChklHU was deposited with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MX 20852 on date 27th August 1997 under Accession No.

ATCC 98520. The full length cDNA and deduced amino acid sequences of human Chkl (ChklHu) are provided in SEQ ID NOs. : 2 and 4 respectively. The full length DNA and deduced amino acid sequences of human Chkl (ChklHu) are provided in SEQ LD NOs.: 1 and 2, respectively.

B. Isolation of a Murine Chkl cDNA

The ChklMo was identified by library screening of a mouse testis cDNA library using a degenerate human Chkl specific probe. The partial cDNA and deduced amino acid sequences of mouse Chkl (ChklMu) are provided in SEQ ID NO.: 3 and 4, respectively.

C. Structural Analysis of ChklHu and ChklMu

The ChklHu and ChklMu cDNAs encode a protein of approximately 70 kD. The kinase domain of ChklHu is 161 to 264 of SEQ ID NO.: 2. The kinase domain of ChklMu comprises amino acids 1 to 61 of SEQ LD NO.: 4. The protein kinase domains of human and mouse Chkl as disclosed herein and are approximately 90% identical at the amino acid level. Relative to C. elegans, S. pombe, and S. cerevisiae Chkl-like proteins, the human form is approximately 56%, 47%, and 37% identical to the protein kinase domains, respectively. Figure 1 compares the amino acid sequences of Chkl homologs from human, mouse, C. elegans, S. pombe and S. cerevisiae. In Figure 1, amino acid residues identical among all species are boxed. Roman numerals indicate subdomains conserved among the homologs. Subdomains V through IX comprise the substrate recognition site and contain the greatest frequency of conserved residues. This is determined by homology with other known kinases (Hanks et al., Science 241 :42-52, 1988).

D. Isolation of a Murine Chkl Genomic Clone

A mouse genomic clone encoding Chkl was obtained using PCR screening of a PI mouse 129 library. The PCR primers used to screen the mouse 129 pi library were mmChk2 (ACG TGG ACA AAC TGG TTC AGG) (SEQ ID NO. : 5) and mmChk 21: CTG ATA GCC CAA CTT CTC GAA SEQ LD NO.: 6). These primers were used to generate an amplicon of 208 bp corresponding to nucleotide X to X of SEQ LD NO.: 4. The amplicon was used to identify a clone of approximately 81-100 kb. An EcoRl restriction digest of the genomic clone was performed and the restriction products were subcloned into a vector with zeocin as the selectable marker. The vector was obtained from Invitrogen p. zero 1.1 (2.8 kb).

The Eco RI restriction digests were resolved on a 0.8% agarose gel and transferred to nitrocellulose according to standard procedures. The nitrocellulose blot was probed with a 208bp amplicon to confirm that a genomic clone was identified.

E. Chromosomal Mapping of the Human Chkl Gene

The Stanford G3 Radiation Hybrid panel was used to map the location of Chkl. PCR reactions using two oligonucleotides in the 3' untranslated region of the human cDNA library derived Chkl DNA fragment yielded a unique PCR amplicon with primer l(Chkl 30mer3'UT) GGCTCTGGGGAATCCTGGTGAATATAGTGCTGC (SEQ ID NO.: 7 and primer 2 (Chkl 30mer 3'UT)

TCCCCTGAAACTTGGTTTCCACCAGATGAG (SEQ LD NO. 8. For sublocalization, chromosome 11 radiation hybrid DNA samples obtained from Research Genetics (Huntsville, Alabama) were analyzed and the results were decoded by the RH server at http://shgc.stanford.edu/. ChklHu localized to marker Dl 154610 which has been mapped to the telomeric region of 11 q at 23.3. This region has been identified as the site of tumor suppressor genes implicated in ovarian, breast, lung, colon and cervix cancer and melanoma. [Gabra et al., Cancer Research, 56:950-954 (1996)].

Example 2 Chkl was expressed in recombinant host cells.

A. Expression and Kinase Activity of Chkl Glutathione-S-Transferase Fusion Protein

DNA encoding Chkl glutathione-S-transferase fusion protein (GST-ChklHu) was also prepared. A DNA encoding GST-ChklHu was cloned into pGEX KGH using Ndel Sail restriction sites introduced into ChklHu. The internal Nde I sites were eliminated by silent mutagenesis. E. coli F Dh5a transformed with GST-Chkl were used to prepare recombinant proteins. To 200 ml of culture, 5mM LPTG was added and cells were grown for four hours at 37 C. The culture was harvested and washed in STE buffer (lOmM Tris pH 8, 150 mM NaCl, and lmM EDTA). The cells were resuspended in 6 mol STE with lmM PMSF and lOOmg lysozyme. The cultures were incubated on ice for fifteen minutes, and 5 mM DTT and 1.5% Sarkosyl was added and the cells were sonicated. The debris was pelleted, and the supernatant was made to 2% Triton X-100. Glutathione agarose beads were added and the beads were pelleted in a benchtop centrifuge and bound proteins were eluted with elution buffer (50mM Tris pH

8.0, 15 0 mM NaCl, lmM PMSF, and 10 mM glutathione.

Kinase assays were performed by incubating GST-ChklHu in kinase buffer (25M HEPES, pH 7.7; 50 mM KC1; 10 mM MgC12; 0.1% NP-40; 2% glycerol; lmM DTT, 50uM ATP) with or without lmg os substrate protein, and incubated in kinase buffer containing lOmCi [g-32P] ATP (3000 Ci mmol) for twenty minutes at 37 C. The reactions were stopped with 20 ul 2 X SDS sample buffer prior to separation on 6% PAGE. Kinase reactions were then transferred to Immobilon, exposed to film, then subsequently probed to detect precipitated protein.

The glutathione affinity purified fusion was able to autophosphorylate and phosphorylate substrate proteins such a s myeline basic protein showing that Chkl is active as a protein kinase, independent of regulatory subunits. Example 3

Antibodies specific for mammalian Chkl proteins were generated as follows.

A. Generation of Polyclonal Antibodies

Polyclonal antibodies were generated against a mouse Chkl polypeptide fragment. CLKETFEKLGYQWKK (amino acids 191 to 203 of SEQ ID NO.: 3) was coupled to Keyhole Lympet Hemocyanin (KLH) via the N-terminally added cysteine and a rabbit was injected with 150 mg per injection. To affinity purify the rabbit sera, 3 mis of thiol coupling gel (TCGel Quality Controlled Biochemicals) (St. Louse, Missouri) that was equilibrated with degassed TEB (50 mM Tris, 5 mM EDTA-Na, pH 8.5) was mixed with 1.25 mg of HPLC purified peptide. The coupled resin was loaded into an econo column (Bio Rad) (Hercules, California) and was washed with 10 column volumes of TEB. The resin was treated blocking buffer (50 mM cysteine in TEB buffer per ml of gel) and was sequentially washed with 20 column volumes of salt buffer. The column was washed with 20 column volumes of salt buffer (500 mM NaCI in 50 mM NaH2PO4, pH 6.5) and 10 column volumes of phosphate buffer (50 mM NaH2PO4, pH 6.5 ). Twenty mis of rabbit serum was loaded onto the column and was washed with 10 column volumes of salt buffer and the antibody was eluted with glycine buffer (lOOmM Glycine-HCL, pH 2.5). One ml fractions were collected in 50 ul of 1.0M Tris, (pH 9.5). Fractions containing antibody were pooled and dialyzed in storage buffer (lOmM NaH2PO4, 20mM MgCl, pH 7.0) and stored at -20 as a Chkl #2-3. The polyclonal antisera, a Chkl #2-3 was able to immunoprecipitate mouse Chkl protein from mouse testes extract as described in Example 6. In addition, aChkl#2-3 recognized recombinant human Chkl and the ChklHu glutathione-S-transferase fusion protein expressed in E. coli.

B. Generation of Monoclonal Antibodies

Monoclonal antibodies are prepared by immunizing Balb/c mice subcutaneously with Chkl, Gst-Chkl or a Chkl fragment in complete Freund's adjuvant (CFA). Subsequent immunizations in CFA or incomplete Freund's adjuvant is performed to increase immune response. The spleen of the immunized animal is removed aseptically and a single-cell suspension is formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension is filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, New Jersey), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI. Thymocytes taken from naive Balb/c mice are prepared in the same manner.

Two x 108 spleen cells are combined with 4 x 107 NS-1 cells (kept in log phase in RPMI with 11% fetal bovine serum (FBS) for three days prior to fusion), centrifuged and the supernatant is aspirated. The cell pellet is dislodged and 2 ml of 37 C PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer Mannheim) is added while stirring over the course of one minute, followed by the addition of 14 ml of serum free RPMI over seven minutes. Additional RPMI can be added and the cells are centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 mM sodium hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5 x 106 thymocytes/ml. The suspension is dispensed into ten 96-well flat bottom tissue culture plates (Corning, United Kingdom) at 200 ml/well. Cells are fed on days 2, 4, and 6 days post-fusion by aspirating 100 ml from each well with an 18 G needle (Becton Dickinson), and adding 100 ml/well plating medium containing 10 U/ml IL-6 and lacking thymocytes.

When cell growth reaches 60-80% confluence (day 8-10), culture supernatants are taken from each well and screened for reactivity to Chkl by ELISA.

ELISAs are performed as follows. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) are coated at 4 C with 50 ml/well with lOOng/well of Chkl in 50 mM carbonate buffer, pH 9.6. Plates are washed with PBS with 0.05%, Tween 20 (PBST) and blocked 30 minutes at 37 C with 0.5% Fish Skin Gelatin. Plates are washed as described above and 50 ml culture supernatant is added. After incubation at 37 C for 30 minutes, 50 ml of horseradish peroxidase conjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) [diluted 1 : 10,000 in PBST] is added.

Plates are incubated at 37 C for 30 minutes, washed with PBST and 100 ml of substrate, consisting of 1 mg/ml TMB (Sigma) and 0.15ml/ml 30% H2O2 in 100 mM Citrate, pH 4.5, is added. The color reaction is stopped with the addition of 50 ml of 15% H2SO4. A450 is read on a plate reader (Dynatech).

Example 4

Northern analysis was performed to determine tissue distribution of Chkl in mouse and human tissue. Oligonucleotides mmchkl :

GTTGAGACTCCATCATCAAGG (SEQ ID NO.: 9) and mmChkl': TCTGGCTGGGAACTAGAGAAC (SEQ ID NO.: 10) were used to generate an amplicon of 220bp, identified as mmChkl+1'. mmChkl+1' and mmChk2+2' (Example 1) were used as probes for northern analysis. The conditions for PCR were as follows: One cycle of eight minutes at 94 °C then forty cycles of 94 °C for twenty seconds, 58 °C for twenty seconds, 72 °C for twenty seconds. The products were analyzed on a 4% agarose gel. The amplicons were labeled with 32P-ATP for Northern analysis. A nylon membrane containing 2mg of size fractionated poly (A)+ RNA from human and mouse tissue sources including human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes, and mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Clonetech Laboratories, Palo Alto, California) was probed with the labeled amplicons as recommended by the manufacturer except that the final wash was performed at 55° C to minimize the possibility of cross-hybridization to related sequences.

In mouse tissue, Chkl expression was observed in lung, spleen, and testes in the mouse. In human tissue, expression was seen in thymus, lung, prostate, and testes. However, testes RNA samples from both mouse and human show approximately two to four fold higher levels of RNA expression than other tissues.

To determine Chkl expression in developing mouse embryos, mRNA obtained from total embryos at day 7, 11, 15, and 17 were probed with labeled mChkl+1' and mmChk2+2'. The Northern blots showed that Chkl expression peaked at day 11 of embryogenesis. Example 5 Because of the roles Atr and Atm play in meiosis and their association with meiotic chromosomes, the expression of ChklMo protein was examined by immunohistological characterization of cross-sections of mouse testes. In testes, pre-meiotic cells surround the surface of the seminiferous tubule and progression towards the interior lumen of the tubule corresponds to progressively later stages of meiosis and sperm maturation [Moens et al., J. Histochem. Cytochem, 25:480 (1977)].

Testes from normal and mutant mice (discussed infra) as described in Heiner et al., Cancer Res., 57: 1664-1667 (1997) were obtained and preserved in Tissue Tek OCT compound, a tissue freezing media, containing 10.24% w/w polyvinyl alcohol,

4.26% w/w polyethylene glycol, and 85.50% w/w nonreacting ingredients. Both wildtype and mutant mice were tested and analyzed by immunohistochemistry using aChkl#2-3, a polyclonal antibody. Six micron cryosections were placed in a 50 C oven to dry and fixed in 4 C acetone for two minutes. The slides were incubated with aChkl#2-3 antisera at a 1 : 100 dilution for thirty minutes. The secondary, goat anti-rabbit biotinylated antibody was applied to each section at a dilution of 1 :200 and incubated for fifteen minutes at 37 C. The tertiary antibody, goat anti biotin was applied to each section at a dilution of 1 :200 and incubated for fifteen minutes at 37 C. After each incubation, the slides were rinsed with 1 X TBS. DAB horseradish-peroxidase substrate was used to detect positive signal in the samples. The reaction was stopped in water and counterstained with Gill's Hematoxylin.

The testes of ChklMo, atm+/+ p53+/+ (A), atm+/+ p53+/- (B) atm-/- p53+/+ (C), atm-/- p53+/- (D), and, atm-/- p53-/- (E) were immunohistologically characterized using anti-Chkl antiserum. Testes cryosections were stained with affinity-purified anti-Chkl antiserum (aChkl #2-3) and with an anti-ATR monoclonal

(224C). The specificity of staining was confirmed by examining pre-immune serum and by specific and non-specific peptide block experiments. Affinity purified Chkl polyclonal antisera (Chkl #2-3 of Example 3) was used to determine the localization.

In contrast to staining patterns reported for Atr, ChklMo shows temporal increases and decreases in nuclear staining in normal mice. ChklMo is most highly expressed at pachynema in primary spermatocytes, indicating that ChklMo may act downstream of Atr function during meiotic prophase, after Atr, which acts earlier during zygonema. Since Atm and p53 have checkpoint properties and may act at an earlier phase in signaling relative to ChklHu/Mo, the localization of ChklMo by histological analyzes was examined in atm-/-p53+/+, atm-/-p53-/- and atm-/-p53+/- mice [Done hower et al., Nature, 356:215 (1992); Kuerbitz et al., Proc. Natl. Acad. Sci. USA, 89:7491 (1992);

Westphal et al., Nat. Gen., 16:397-401 (1997)]. ChklMo accumulation and localization was independent of p53 status but dependent on Atm, suggesting that ChklMo also acts downstream of Atm in meiotic prophase.

To further analyze the role of Chkl in mammalian meiosis, the temporal and spatial distribution of Chkl in surface spread preparations of spermatocytes was determined.

For the meiotic preparations, surface spreads of spermatocytes from fifteen to twenty-one day old mice (C57-bl/6) were prepared and antibody incubation and detection procedures were preformed as described in Ashley et al., Chromosoma, 104: 19 (1995). Antibody incubation and detection procedures were a modification of the protocol of Moens et al. as described previously in Ashley et al. Since the antibodies against Chkl and Sep3 (control) were both raised in rabbit, spermatocytes were labeled and imaged sequentially. Goat-anti rabbit IgG-rhodamine-conjugated and goat-anti rabbit IgG-FITC-conjugated (Pierce) secondary antibodies were used for detection. All preparations were counterstained with 4', 6' diamino-2-phenylindole (DPAI, Sigma) and mounted in a DABCO (Sigma) antifade solution. The preparations were examined on a Zeiss Axioskop (63-X and 100-X, 1.2 Plan Neoflour oil-immersion objective). Each fluorochrome (FITC, rhodamine and DAPI) image was captured separately as an 8-bit source image using a computer assisted cooled CCD camera (Photometries CH220) and the separate images 24-bit pseudocolored and merged with custom software developed by Tim Rand [Ried et al., Proc. Natl. Acad. Sci. USA, 89:1388 (1992)].

Zygotene spermatocytes were stained with an antiserum against Scp3, a component of the axial element, which forms between the sister-chromatids. The same zygotene spermatocytes were labeled with a-Chkl#23. Chkl is present along the synaptonemal complexes (SC) of synapsed homologous chromosomes. Chromosomes that are in the process of synapsing have Chkl staining, but no staining is observed on the unsynapsed axial elements As meiosis proceeds into pachynema, ChklMo remains associated with autosomal synaptonemal complexes in a focal staining pattern similar to ATM

A pachytene spermatocyte was labeled with both anti-Chkl and a mouse monoclonal antibody against Atr (224C) Although Chkl initially appears in a focal pattern, in pachynema Chkl seems to accumulate along the SCs In addition, Chkl foci appear along the unsynapsed axial elements of the X and Y chromosomes in mid pachynema where it colocalizes with Atr Chkl remains on the SCs throughout pachynema and disappears when the homologous chromosomes disassociate in diplonema Progression of meiotic prophase I appears to be normal in p53-/- mice In an early pachytene spermatocyte labeled for Chkl and Scp3, Chkl is present along the SCs and the synapsed region of the sex chromosomes However, Chkl does not appear along the unsynapsed axial element of the X chromosome in early pachytene In atm-/- mice, progression of meiosis is disrupted as the SCs begin to fragment following synapsis Although homologous chromosomes synapse in atm-/- spermatocytes, no Chkl is detected along the synapsed bivalents or fragmented SCs

Thus, in normal mice, Chkl appears in a focal pattern along the synaptonemal complexes of synapsing homologous chromosomes in zygonema, in a pattern similar to that of Atm Chkl accumulates along the SCs as meiosis progresses into pachynema, and by mid-pachynema Chkl coats the entire SCs In early pachynema,

Chkl is present along the synapsed region of the XY bivalent However, by mid-pachynema, Chkl foci also appear along the unsynapsed axes of both the X and Y chromosomes, where it colocalizes with Atr Atr is found in foci along the unsynapsed axes of the sex chromosomes early, and later coats the entire X and Y axes throughout pachynema

Meiosis appears to be unaffected in p53 deficient mice, as is demonstrated by histological analysis and immuno staining of surface spread spermatocytes with Scp3 and Chkl In contrast, atm-/- mice are sterile as the result of progressive fragmentation of meiotic chromosomes following synapsis, leading to apoptosis Immunolocalization of Chkl in atm-/- spermatocytes indicates a lack of Chkl on the SCs, suggesting a role of Chkl downstream of Atm in mammalian meiosis To determine if the lack of Chkl staining in atm-/- nuclei and the lack of ChklMo protein on meiotic prophase chromatin was reflected by the level of ChklMo, Western analysis of testes extracts was performed according to Keegan et al., Genes Dev., 10:2423 (1996). Chkl protein was present in an Atm-dependent fashion, suggesting that synthesis or stability of ChklMo depends on the Atm protein. MTE of mice lacking Atm did not stain for Chkl indicating that atm-1- mice did not express Chkl . In contrast, MTE of wild type mouse or from mice in which p53 expression was disrupted showed Chkl staining.

Example 6 The kinase activity of mouse Chkl and its ability to associate with Atr were also demonstrated.

A. Kinase Activity of Murine Testes Chkl

Antibody aChkl#2-3 also used to immunoprecipitate Chkl from mouse testes extract (MTE). Approximately 30 decapsulated testes were ground in a mortar with liquid nitrogen and the grounds were transferred to a 15 ml dounce homogenizer. Fifteen mis of lysis buffer (50mM NaPO4, pH 7.2, 0.5% TritonX-100, 2mM EDTA, 2mM EGTA, 25mM NaF, 25mM 2-glycerophosphate, lmM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml. leupeptin, 1 mg/ml pepstatin A, and 2mM DTT) was added and the extract was dounced 30 times with a loose pestle and 20 times with a tight pestle. After a low speed spin, the supernatant assayed using BCA for protein concentration determination.

For Chkl immunoprecipitations, 400mg of MTE extract was incubated with either lOmg of affinity purified Chkl #2-3 or with approximately lOmg of preimmune serum for thirty minutes on ice. Protein A-agarose slurry (Pierce) was added and the mixture was incubated for thirty minutes at 4 C. The immune-complex bound slurry was washed three times in TSAT and one time with kinase buffer (25uM HEPES, pH 7.7; 50 mM KC1; 10 mM MgC12; 0.1% NP-40; 2% glycerol; lmM DTT, 50uM ATP), and incubated in kinase buffer containing lOmCi [g-32P] ATP (3000 Ci mmol) for twenty minutes at 37 C. The reactions were stopped with 20 ul 2 X SDS sample buffer prior to separation on 6% PAGE. Kinase reactions were then transferred to Immobilon, exposed to film, then subsequently probed to detect precipitated protein.

To determine if the immunoprecipitated Chkl from MTE could self-phosphorylate, 2X kinase buffer and 10 mCi g32-P-data (3000 Ci/mM) were added to the immunoprecipitate. The phosphorylation reactions were incubated at 30 C for fifteen minutes. The reactions were electrophoresed on a 6% PAGE gel, transferred to immobilon P, and exposed to X-ray film. The blots showed that immunoprecipitated Chkl was able to self phosphorylate as was the Chkl -GST fusion protein (Example 2). Mouse IgG and Chkl preimmune sera did not immunoprecipitate ChklMo.

C. Association of Chkl and Atr

To determine if Chkl and Atr can associate in meiotic cells, 460 mg of MTE was immunoprecipitated with anti-Atr monoclonal antibody (aAtr-224C) under conditions as described above. The Atr immunoprecipitate was electrophoresed on a 6% or 8% PAGE, electroblotted onto immoblin P membrane and was probed with anti-Chkl antibody (aChkl #2-3). The blots showed that Chkl co-precipitates with Atr indicating that Atr and Chkl associate in meiotic cells. In addition, the Chkl that immunoprecipitates with Atr was able to self phosphorylate.

Numerous modifications and variations in the practice of this invention are expected to occur to those of skill in the art. Only such limitations that appear in the appended claims should be placed on the invention.

Claims

CLAIMSWe claim:

1. A purified and isolated polynucleotide encoding the human Chkl kinase amino acid sequence set out in SEQ ID NO: 2.

2. A purified and isolated polynucleotide encoding the mouse Chkl kinase amino acid sequence set out in SEQ ID NO.: 4.

3. The polynucleotide of claim 1 or 2 which is a DNA.

4. The DNA of claim 3 which is a cDNA.

5. The DNA of claim 3 which is a genomic DNA.

6. The DNA of claim 3 which is a wholly or partially chemically synthesized DNA.

7. A human Chkl DNA comprising the DNA sequence set out in SEQ LD

NO.: 1.

8. A mouse Chkl DNA comprising the DNA sequence set out in SEQ ID NO.: 3.

9. An RNA transcript of the DNA of claim 3.

10. A DNA encoding a full length mammalian Chkl kinase selected from the group consisting of: a) a DNA which hybridizes under stringent conditions to the non-coding strand of the DNA of SEQ ID NO.: 2; and b) a DNA which hybridizes under stringent conditions to the non-coding strand of the DNA of SEQ LD NO.: 4.

11. A vector comprising a DNA according to claims 1, 2, 3 or 10.

12. The vector of claim 11 wherein said DNA is operatively linked to an expression control DNA sequence.

13. A host cell stably transformed or transfected with a DNA according to claims 1, 2, 3 or 10 in a manner allowing the expression in said host cell of the Chkl kinase.

14. A method for producing Chkl kinase, said method comprising growing a host cell according to claim 11 in a suitable nutrient medium and isolating the Chkl kinase.

15. A purified and isolated polypeptide comprising the human Chkl kinase amino acid sequence consisting of SEQ LD NO.: 2.

16. A purified and isolated polypeptide comprising the mouse Chkl kinase amino acid sequence consisting of SEQ ID NO.: 4.

17. A polypeptide or peptide capable of specifically binding to mammalian Chkl kinase.

18. The polypeptide according to claim 17 which is an antibody.

19. The antibody according to claim 18 which is a monoclonal antibody.

20. A hybridoma cell line producing the monoclonal antibody according to claim 19.

21. A method of identifying a compound that is a modulator of mammalian Chkl kinase comprising the steps of: a) determining the kinase activity of Chkl in the presence and absence of said compound; b) comparing the kinase activities observed in step (a); and c) identifying said compound as a modulator by the observed differences in the kinase activity of Chkl in the presence and absence of said compound.

22. A method of identifying a compound that inhibits mammalian Chkl comprising the steps of: a) expressing mammalian Chkl in a genetically altered cell, thereby decreasing the sensitivity of the cell to DNA damage, said sensitivity being associated with the genetic alteration; b) exposing the genetically altered cell of step (a) to DNA damaging treatment in the presence and absence of a test modulator compound; c) measuring the sensitivity of the cell to DNA damage; and d) identifying a test compound that restores the sensitivity of the cell to DNA damage as an inhibitor of Chkl activity.