Abstract of the funded parent award Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to eliminate DNA double-strand breaks and chromosomal lesions. Mutations in HR mediators dictate the pathological progression of many cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and protected by Replication Protein A (RPA). The Breast Cancer Type 2 Susceptibility (BRCA2) protein functions to remove and remodel RPA and promote binding of the RAD51 recombinase, which then catalyzes strand exchange and drives recombination. Our long-term goals are to gain a mechanistic understanding of the temporal sequence of ssDNA handoff between these HR mediators and how these mediators compete for access to ssDNA and its overall contribution to diseases such as cancer. RPA is composed of multiple distinct DNA binding domains (DBDs) and binds with high affinity to ssDNA. The intrinsic DNA binding dynamics (binding, dissociation and remodeling) of individual DBDs are hypothesized to dictate when, where, and how RPA functions. For several decades the four DBDs of RPA have been classified as high affinity (DBDs-A & B) and low affinity (DBDs-C & D) based on experimental measurements of ssDNA binding affinity of isolated DBDs and have shaped models for RPA mechanism in DNA replication, repair and recombination. Using non-canonical amino acids, we developed a fluorescence-based method to capture the dynamics of individual DBDs in the context of the full-length protein. In contrary to classical models, we uncovered that the high-affinity DBDs are dynamic and are outcompeted by the trimerization core of RPA. In lieu of these exciting discoveries of RPA mechanism, we here propose to uncover how the BRCA2 mediator influences the dynamics of RPA-DBDs to promote the formation of the RAD51 filament during HR. We will establish how the context and type of DNA structures that are encountered in HR affect RPA-DBD dynamics [Aim 1]. We will investigate the importance of a regulatory hotspot in RPA that would modulate the interaction between RPA and the BRCA2-DSS1 complex [Aim 2]. Finally, using fluorescent versions of BRCA2, RAD51 and RAD52, we will establish the mechanism of ssDNA handoff from RPA to RAD51 during HR [Aim 3].

NARRATIVE Defects in homologous recombination (HR) are associated with genomic instability and rearrangements resulting in a variety of cancers and inherited disorders. The proposed studies will delineate the mechanism by which cells process double-stranded breaks in DNA through HR. The results from our research will have a direct relevance to understanding how mutations in HR enzymes cause cancers and better direct therapeutic interventions.