) Patients with ataxia telangiectasia (A-T) are predisposed to cancers of different origins. The gene mutated in ataxia telangiectasia, ATM, is required for normal cellular responses to DNA double-strand breaks (DSBs). A-T cells are not only defective in cell cycle checkpoints but also show abnormalities in DSB repair. We hypothesize that ATM-mediated signals guard genome integrity and prevent cancer formation by modulating the assembly and function of DSB repair protein complexes. This hypothesis is based upon our discovery of a novel ATM/c-Abl signaling pathway that mediates the phosphorylation of Rad51 protein, which results in an enhancement of its interaction with Rad52. Rad51, the eukaryotic equivalent of the E. coli recombination protein RecA, and Rad52 are both required for homologous recombination and the recombinational repair of DSBs. To further characterize the dynamic repair processes in cellular responses to DNA damage: (1) We will study the ATM kinase activity with a major emphasis on identifying protein substrates involved in the repair of DSBs. (2) We will map phosphorylation sites in Rad51 and in Rad50, a component of the Mre11/Rad50/p95 nuclease complexes that process DSBs for recombinational repair. (3) The manner in which ATM-dependent phosphorylation of Rad51, Rad50, and other repair factors affects the assembly of protein complexes and the biochemical activities of these proteins will be studied in vitro, and also in vivo using A-T, NBS cells harboring mutation in ATM and p95 respectively, and mouse embryonic fibroblasts established from wild-type, ATM, and c-Abl knockout mice. (4) To assess the role of ATM-mediated phosphorylation in cancer predisposition, we will perform mammary gland transplantation using wild-type, ATM heterozygous and homozygous tissues. We will establish transgenic mice expressing Rad51 phosphorylation mutants. IR-induced responses will be studied using these models. The incidence and/or progression of cancers in these animals will be determined. These studies will be conducted in close collaboration with the investigators in Projects 1, 2, and 4, and will utilize the macromolecule synthesis and analysis, imaging, and animal cores.
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