PROJECT SUMMARY Despite several years of investigation, the human RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 remain enigmatic proteins required for cell viability and homology-directed repair (HDR) of DNA double-strand breaks (DSBs). The RAD51 paralogs have been found to exist in two protein complexes within human cells: RAD51B/RAD51C/RAD51D/XRCC2 and RAD51C/XRCC3. Our preliminary data indicate that the RAD51 paralogs interact with BRCA2 in a specific orientation likely important for mechanistic control over the RAD51 nucleoprotein filament. Our objective in this proposal is to address the biochemical and genetic relationship between BRCA2 and the RAD51 paralogs in response to DNA damage. Our hypothesis is that BRCA2 and the RAD51 paralogs work together in the pre- or post-synaptic phase of HDR to either enhance RAD51 nucleoprotein filament stability or to stimulate strand invasion and the homology search. We will map the sites of interaction between BRCA2 and the RAD51 paralogs. We will determine whether interactions are regulated by DNA damage and what impact the BRCA2/RAD51 paralog complex has on RAD51 filament dynamics. We have developed human cell systems from which to purify the RAD51 paralog proteins individually or in complexes (B/C/D/X2 and C/X3). These purified proteins will then be used for biochemical studies of HDR. BRCA2 has been linked to stabilization of replication forks to prevent nucleolytic degradation under conditions of cellular stress, and therefore, we will determine whether the RAD51 paralogs cooperate with BRCA2 to stabilize RAD51 at stalled replication forks through analyses of DNA fibers and super-resolution microscopy. In summary, we plan to use both biochemical and genetic approaches to understand the interplay between BRCA2 and the RAD51 paralogs and how defects in these proteins lead to genomic instability and cancer.