The goal of the proposed research is to determine the structural and enzymatic roles of several mammalian proteins in homologous recombinational repair (HR) of DNA double-strand breaks (DSBs), and to determine how these proteins regulate spontaneous HR. DSBs are induced by chemicals and radiation and they arise spontaneously during DNA replication. DSBs are repaired by HR and by nonhomologous end-joining (NHEJ). Proteins with direct roles in HR include RAD51, five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D), BRCA1, and BRCA2. RAD51 catalyzes DNA strand transfer and is therefore central to HR processes. However, RAD51 is difficult to study in mammalian cells because null mutants are cell- and embryo-lethal. The RAD51 paralogs form several complexes, and these complexes physically interact with RAD51 and/or DNA, and are thought to augment RAD51 activity. BRCA2 interacts with RAD51 and other proteins, as well as single- and double-stranded DNA. The RAD51 paralogs and BRCA2 are thus uniquely positioned to regulate HR efficiency and outcome. Defects and/or altered expression of any of these proteins result in genome instability and cancer predisposition. We recently demonstrated that XRCC3 influences early and late stages of HR, and that several BRCA2 functional domains have dominant negative effects on DSB-induced HR in human cells. Two specific aims are proposed to investigate (1) the structural and enzymatic roles of XRCC2, XRCC3, and RAD51C in DSB-induced and spontaneous HR; and (2) the roles of several functional domains of BRCA2 in DSB-induced and spontaneous HR, including BRCA2 domains that interact with single- and double-stranded DNA, RAD51, and other proteins. These projects will help establish roles for specific structural or enzymatic domains of XRCC2, XRCC3, RAD51C, and BRCA2 in early and/or late stages of HR, and thereby provide new insight into the regulation of HR, maintenance of genome stability, and tumor suppression. Because DSB repair is a key determinant of cell survival following DNA damage, the information gained from these studies will also facilitate the development of more effective agents to sensitize tumor cells to DNA damage by radiation and genotoxic chemicals. Studies of HR mechanisms and HR regulatory proteins will also facilitate the development of more effective gene targeting and gene therapy systems.