When cells are exposed to physical or chemical agents that damage DNA, deleterious effects can ensue, including mutation, cancer or death. However, mechanisms are available to repair the damage and neutralize harmful effects. During the previous funding period, focus was placed on understanding the role of human HRAD9 in processes that promote survival and genomic integrity after radiation or chemical exposure. Our studies indicate that this evolutionarily conserved gene has multiple functions needed for the cellular response to DNA damage. We found that HRAD9 has a BH3-like domain in its N-terminal region, is capable of binding anti-apoptotic proteins BCL-2 and BCL-XL, and can induce apoptosis in a BH3- dependent manner. We also found that cell cycle checkpoint control proteins HHUS1 and HRAD1 can bind the C-terminal region of HRAD9, suggesting that the protein has at least two functional domains. In addition, we found that ATM can phosphorylate HRAD9, an event important for G1/S checkpoint control. Recently, we obtained evidence that HRAD9 has two other activities, which are the focus of this proposal. HRAD9 can physically associate with p53 and, like p53, it can act as a sequence specific transcription factor. HRAD9 can bind p53 consensus sequences in the p21 promoter and cause transcription. In addition, HRAD9 can bind the recombination protein HsRAD51, and our initial data suggest that HRAD9 also plays a role in homologous recombination. These two novel HRAD9 activities will be pursued by addressing the following two hypotheses: 1. HRAD9 is a sequence specific transcription factor that, like p53, can regulate the cellular response to DNA damage by transactivation of multiple genes-i.e., it controls a novel damage response regulon. Experimental aims concern structural requirements of the activity, and the identification and biological significance of inducible genes in the network. 2. HRAD9 participates in homologous recombination and possibly nonhomologous end joining, two processes important for repair of dsDNA breaks. Experiments are proposed to establish the function of HRAD9 in these processes. The results of this study should better define the role of HRAD9 in the cellular response to DNA damage, in particular as a novel regulator of damage-inducible genes and a participant in recombinational repair. As such, the findings should impact on basic research and clinical arenas where DNA damaging agents are used for therapy.
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