Cell-cycle checkpoints are signal transduction pathways that ensure orderly progression of the cell cycle, and that proliferating cells do not attempt to replicate or segregate damaged chromosomes. Recent studies have pinpointed two members of the P1 3-kinase related kinase family, ATM and ATR, as proximal signaling elements in the G1, S, and G2 checkpoint pathways. The emerging view is that ATM function is required for the induction of appropriate checkpoint responses to ionizing radiation-induced DNA double-strand breaks, while ATR plays dominant roles in the responses to ultraviolet light induced DNA damage, as well as to DNA replicative stress induced by drugs or intrinsic errors associated with DNA metabolism. In spite of this progress, the mechanisms underlying the regulation and function of ATR remain unclear, due in large part to the lack of a genetically tractable, ATR-deficient cellular model system. Similarly, although our preliminary studies have identified two important genome surveillance proteins, BRCA1 and hRad17, as substrates for the ATM/ATR kinases in DNA-damaged cells, the functional consequences of these phosphorylation events have not been defined. This project will address these critical issues through implementation of the following specific aims: (1) To complete the generation of a novel ATR-/- cell line, and to characterize the checkpoint signaling defects in these cells, and (2) To determine the impact of ATR-mediated phosphorylation on the genome maintenance functions of BRCA1, through the use of two newly described functional assays for BRCA1, (3) To define the regulatory effects of ATR/ATM-dependent phosphorylation on the checkpoint signaling functions of hRad17, and (4) To identify and functionally characterize novel SIT-Q-containing substrates for ATR and ATM in cells exposed to genotoxic stress.
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