ATM, the gene product mutated in ataxia-telangiectasia (A-T), is the master regulator of the cellular responses to DNA double-stranded break (DSB) damage. As a protein kinase, ATM phosphorylates and activates several tumor suppressors, including p53 and NBS1, after DNA DSB damage. Similar cellular and systematic defects observed in ATM and NBS 1 mutant mice provided genetic evidence that large portions of ATM functions are mediated by NBS1. Therefore, to understand the mechanism of pathogenesis in A-T, I propose to study the ATM-dependent as well as ATM-independent functions of NBS1 in animal development and cellular responses to DNA DSB damage. The importance of NBS 1 in the p53 responses to DNA DSB damage remains controversial. Therefore, I propose to employ NBS 1 mutant mice we generated to determine whether NBS1 is required for p53 responses to DNA DSB damage. In addition, since ATM activates both NBS1 and p53 after DNA DSB damage, the functional synergy of NBS1 and p53 in tumor suppression will be tested. H2Ax is important for the repair of DNA DSN damage. Phosphorylation of histone H2AX, namely gamma-H2AX, occurs within minutes after introduction of DNA DSBs and ATM family kinases are the major kinases to mediate gamma-H2AX after DNA damage. Therefore, gamma -H2AX could function as a mediator directly downstream of ATM to signal cellular responses to DNA DSB damage. To determine the physiological roles of gamma-H2AX in the cellular responses to DNA damage, I propose to employ homologous recombination to introduce the missense Ser139Ala mutation into the endogenous H2AX in mice. H2AX s139A mice will be examined for systemic defects and their cellular responses to DNA damage. These studies should reveal the physiological roles of gamma-H2AX in ATM-dependent as well as ATM-independent cellular responses to DNA damage and tumor suppression. In addition, I propose to investigate the functional interactions between NBS1 and H2AX, the two mediators of ATM functions.
