Project Summary/Abstract The broad objective of this proposal is to understand the molecular mechanism of homologous recombination (HR) in humans, and to understand how the consequences of defects in recombinational DNA repair result in chromosomal instability that underlie the predisposition to cancers. The proteins involved in a central step of HR, DNA pairing, include RAD51, BRCA2, and the RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. Mutations in these proteins are known to predispose individuals to cancer. The objective is to understand the functions of these proteins in genome maintenance via recombinational DNA repair, and how these proteins mediate and potentiate homologous DNA pairing by RAD51. We propose to understand their mechanisms of action through biochemical analyses of reconstituted reactions using purified proteins; characterization of defective mutant proteins identified in the patient population, and by visualizing the individual behavior of these proteins acting on single molecules of DNA. The Specific Aims are to understand the molecular functions of BRCA2 protein and to determine the functions of the RAD51 paralogs in RAD51 filament formation and RAD51-dependent DNA pairing.