The role of BRCA1 and BRCA2 protein complexes in the maintenance of genome stability

Homologous recombination (HR) is an essential and generally accurate pathway for repairing DNA double-strand breaks (DSBs)4. The BRCA1 and BRCA2 tumor suppressors, which are frequently mutated in breast and ovarian cancers, function during HR and DNA replication, yet they remain only partly understood. This research proposal aims to enhance our knowledge on the mechanism of the BRCA1/BRCA2 protein complexes' functions in DNA metabolism through innovative biochemical reconstitution assays.

HR begins with DNA end resection, generating 3' single-stranded DNA (ssDNA) overhangs that become coated by the ssDNA binding protein RPA. These overhangs are subsequently bound by RAD51, which then catalyzes DNA strand invasion into homologous DNA, which is used as a repair template in later HR stages. Although previous biochemical assays have provided detailed insights into HR mechanisms, they investigated the individual HR steps (DNA resection, DNA strand invasion) separately, leaving the coordination of these processes largely unexplored. Our recent work demonstrated that the BRCA1 complex directly facilitates DNA resection. The BRCA2 complex is known to promote RAD51 loading, and BRCA1 again supports strand invasion. Cellular evidence indicates that BRCA1 and BRCA2 form a super-complex required for optimal HR efficiency, and its assembly is regulated in a cell-cycle dependent manner. We hypothesize that the BRCA super-complex helps couple DNA end resection with RAD51 loading. In Aim I, we will utilize our newly developed assay that integrates DNA end resection with DNA strand exchange to determine the physiological roles of the BRCA1/2 complexes and their co-factors in coordinating these processes.

Our previous research established that phosphorylation of BRCA1-BARD1 is required to stimulate long-range DNA end resection1; however, the specific phosphorylation sites and the precise mechanisms by which these modifications stimulate resection activity remain unclear. Aim II will employ our unique biochemical setup to define the impact of BRCA1-BARD1 phosphorylation on DNA resection. Prospective mutants identified through biochemical experiments will subsequently be validated using cellular assays.

RAD51 loading on ssDNA needs to be properly balanced to enable efficient HR, but also to prevent illegitimate recombination and hence genome rearrangements. Both excessive and insufficient RAD51 loading negatively impact genome stability. In Aim III, we will focus on FIGNL1 and co-factors that counteract BRCA2 to disassemble RAD51 filaments to limit illegitimate recombination, in order to understand their mechanism of action.

In Aim IV, we will investigate a non-canonical function of the BRCA1/2 complexes, RAD51 and co-factors to protect nascent DNA from degradation under replication stress—a response with a significant clinical relevance. Recent findings from our group and others suggest that RAD51's protective role likely results from its binding to dsDNA. This property was previously overlooked because it is inhibitory for HR, and hence was thought to be physiologically irrelevant. We plan to clarify how various assemblies of BRCA1 and BRCA2 and co-factors regulate RAD51’s dsDNA binding activity and its DNA protective capacity, which regulates tumoral response to certain chemotherapeutic drugs.

Overall, our studies will provide detailed mechanistic insights into BRCA1/BRCA2 tumor suppressor functions and their influence on critical processes ranging from HR to the protection of nascent DNA. These assays will inform us about the roles of BRCA1/2 complexes in DSB repair and their roles in tumor chemoresistance.