DNA repair processes are essential for maintaining genome integrity and preventing mutagenesis and carcinogenesis. Homologous recombinational repair (HRR), in which Rad51 plays a central role, is a major pathway in yeast for repairing double strand breaks (DSBs) induced by ionizing radiation (IR). However, in mammalian cells neither the biochemical mechanism nor the relative contribution of HRR in cellular response to DNA damage is well understood. The human XRCC2 protein is implicated in HRR by the properties of the IR-sensitive XRCC2 mutant irs1 and the finding that is belongs to a human family of Rad51-like proteins. Also, physical interaction of XRCC2 with another Rad51-like protein, Rad51D, is suggested by yeast 2-hybrid analysis. This project will test the hypothesis that XRCC2 participates in HRR through functional association with human Rad51 (HsRad51). Biochemical activities, such as ATPase, of purified XRCC2 will be characterized, and the interaction of XRCC2 with Rad51D will be confirmed. A key question to be addressed is whether XRCC2 or XRCC2-Rad51D complex acts as an accessory factor to promote HsRad51's strand exchange activity. The biological relevance of XRCC2's ATPase and interaction with Rad51D will be evaluated by expressing mutant cDNAs in hamster irs1 cells to test for complementation. By identifying the other proteins that interact with XRCC2 in human extracts, an attempt will made not only to learn whether the Rad51 family is present in a multiprotein complex, but also to isolate novel proteins interacting with XRCC2. The requirement for XRCC2 in IR-induced HsRad51 nuclear focus formation will be characterized in irs1 cells and cellular colocalization of XRCC2 with Rad51 will be examined to define a functional association of XRCC2 with HsRad51. Finally, cell cycle-dependent expression of XRCC2 and the radiosensitivity of synchronized irs1 cells will be determined to measure the quantitative contribution of XRCC2 to HRR, which likely operates only in the S and G2 phases of the cell cylce. This study will lead to a better understanding of the role of XRCC2 in mammalian HRR and the importance of this pathway in maintaining chromosomal stability.
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