The Specific Aims of this grant are directed towards a genetic, molecular, and biochemical understanding of the function of the KARP-1 gene in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. It is now clear that mammalian DNA DSB repair and site-specific recombination pathways share common factors. In particular, the DNA-dependent protein kinase catalytic subunit, Ku86, Ku70, XR-1 and DNA ligase IV gene products all appear to be required for both of these processes. Recently, we have identified a novel gene, KARP-1 (Ku86 Autoantigen Related Protein-1), that is regulated by the p53 and ataxia telangiectasia (ATM) genes, and which also appears to play a critical role in DNA DSB repair. We describe below experiments which should elucidate the molecular and biochemical role(s) of KARP-1 in DNA DSB repair and V(D)J recombination. The importance of identifying and understanding the genes that control human DNA repair is underscored by the existence of a large number of human cancer predisposition syndromes such as ataxia telangiectasia, Fanconi's anemia, human nonpolyposis colon cancer, Li Fraumeni syndrome, xeroderma pigmentosum and breast cancer, where it appears that the underlying molecular defects reside in DNA repair genes. We intend to characterize these important pathways using five lines of experimentation: 1. Characterization of KARP_1-/- human tissue culture cells. 2. Characterization of KARP-1 gene expression. 3. Characterization of genes that interact with KARP-1. 4. Isolation and characterization of a KARP-1 homolog. 5. What is the subcellular localization of KARP-1? The ultimate goal of these studies is to use the KARP-1 gene as a tool to understand the molecular mechanisms of DNA DSB repair.
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