Genetic studies have implicated the tumor suppressor BRCA2 and the FANCD2 protein, a key member of the Fanconi anemia pathway of DNA damage response, in chromosome damage repair by homologous recombination. BRCA2 binds DNA and associates with the RAD51 recombinase and the FANCD2 protein. We hypothesize that these BRCA2-ligand interactions are germane for chromosome damage repair. Consistent with this hypothesis, our preliminary studies have found enhancement of the RAD51 recombinase activity by a polypeptide derived from BRCA2, in a manner that is dependent on both RAD51 and DNA binding by BRCA2. Our research project will continue to decipher the mechanistic bases and consequences of BRCA2-ligand interactions in the context of DNA break and crosslink repair reactions. To accomplish our goal of deciphering the role of the BRCA2 and FANCD2 proteins in chromosome damage repair, molecular studies under three specific aims will be carried out. In Specific Aim 1, we will conduct a detailed molecular characterization of the BRCA2 DNA binding domain by examining DNA binding specificity, examining the role of the three OB folds in DNA substrate engagement, determining the function of the OB2-appended Tower domain in DNA binding specificity, and also assessing the effects of OB fold and Tower mutations biochemically and genetically. Specific Aim 2 focuses on the role of the BRCA2 carboxyl-terminus in RAD51-mediated homologous recombination. Herein, we will construct, purify, and characterize functional polypeptides of BRCA2 that encompass its C-terminal RAD51 binding domain and employ our unique biochemical systems to test the hypothesis that this C-terminal domain helps shepherd RAD51 to the recombination substrate. Specific Aim 3 is designed to test hypotheses concerning modulation of the BRCA2 DNA binding and recombination mediator functions by FANCD2. To achieve this objective, we will assemble complexes of BRCA2-derived polypeptides and FANCD2 and will examine these complexes for DNA binding and functional interactions with Rad51. The significance of the BRCA2-FANCD2 complex will be ascertained by constructing and characterizing BRCA2 mutants that are defective in FANCD2 interaction. Cell-based functonal assays wil be carried out in collaboration with Projects 1, 2 and 3 and Core C.