Annual Report HS02243-01 Maintenance of genome integrity is essential for survival of all organisms, from bacteria to human. In order to maintain genome integrity, a cell must be able to sense DNA damage, repair the DNA damage and at the same time, halt cell cycle at one of several checkpoints to prevent cell division before DNA damage is repaired. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe, SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S-phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. We have identified a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea (HU) treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after HU treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals. One of the substrates of HuCds1 is BRCA1, the tumor suppressor that is mutated in many familial breast cancers. HuCds1 phosphorylates S988 of BRCA1 and regulates its function in DNA damage repair. This was the first evidence that HuCds1 is important not only for cell cycle checkpoint for but DNA damage response as well. Cds1 family of kinases have a conserved protein motif called FHA (forkhead-associated). We have determined that the FHA domain is essential for ATM to phosphorylate Thr68 of Cds1 and phosphorylation of Thr68 is essential for activation of Cds1 in response to DNA damage. Thr68, in turn, is essential for phosphorylation of Thr373 and 377 in the activation loop of Cds1. The activation loop phosphorylation is a result of autophosphorylation and is essential for Cds1 activity. In collaboration with Dr. Gary Rathbun at Harvard, we have used the peptide library to determine the optimum consensus sequence for Cds1 substrates.
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