The tumor suppressor BRCA2 is a critical factor in homologous recombination (HR)-dependent DNA repair and disabling mutations lead to increased genome instability and cancer predisposition. The essential function of BRCA2 is to facilitate nucleation of the RAD51-ssDNA filament, a rate-limiting step for the formation of this central HR intermediate. BRCA2 engages in interactions with multiple proteins including post-translational modifiers and other regulators. The long-term goal is to elucidate the mechanism of BRCA2 and its binding partners in HR. This project focuses on one specific aim, the overall structure and architecture of full-length human BRCA2 and its modulation by substrate and binding partners. This Specific Aim is: Specific Aim 1: Structure and function of full-length human BRCA2 protein. DSS1 is a critical BRCA2 interaction partner. DSS1 depletion and DSS1 mutations phenocopy a BRCA2 defect. However, the precise mechanism by which DSS1 functions in BRCA2-mediated HR remains to be determined. Our preliminary data suggest an effect of DSS1 on BRCA2 architecture, and thus DSS1 biochemistry is incorporated as Subaim 1A to integrate functional studies to the structural approach. We will test the hypothesis that DSS1 stabilizes monomeric BRCA2 on ssDNA to nucleate RAD51 filament formation. The possibility of obtaining a crystal structure of full-length human BRCA2 is not feasible at present because of the size and the segmental nature of the protein. We will use TEM and cryo-EM to determine the overall structure of BRCA2 alone and in functional complexes with DNA, DSS1, and RAD51, to elucidate the effects of its substrate and main binding partners on the overall architecture of BRCA2. Preliminary data show that we can visualize full-length human BRCA2, whose identity was confirmed by immunogold-antibody staining and targeting the C-terminal His tag. Upon binding DSS1 and ssDNA, BRCA2 transitions to an open C-shape monomer with a significant conformational change from the monomers or dimers found in solution. Using the extraordinary EM resources of our collaborator Dr. Al-Bassam, we will reconstruct 3D structures of human BRCA2 using TEM imaging (Subaim 1B) and cryo-EM (Subaim 1C). We are fully aware of image reconstruction artifacts, and we safeguard against this by having two independent reconstruction efforts by Drs. Al-Bassam and Stahlberg.