Biochemical and Mechanistic Insights into the Roles of Rad51 Paralogs in Homologous Recombination Repair

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PROJECT SUMMARY/ABSTRACT Understanding the molecular pathways disrupted in breast and ovarian cancer is vital to combating these devastating diseases. Hereditary susceptibility to cancer can be caused by germline mutations in the BReast CAncer susceptibility genes BRCA1 and BRCA2. The BRCA genes code for proteins involved in homologous recombination (HR) repair of DNA Double-Stranded Breaks (DSBs) and the protection of reversed replication forks. BRCA2, in particular, is vital to HR as it loads RAD51 onto single-stranded DNA (ssDNA) forming a nucleoprotein filament. The five RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 are a family of proteins with homology to RAD51, have been implicated in HR regulation, and germline mutations have been linked to familial breast and ovarian cancer. The RAD51 paralogs form two different complexes in human cells but their functions and stoichiometric relationships in HR remain uncharacterized decades after their discovery. The BCDX2 complex (RAD51B/C/D/XRCC2) and the CX3 complex (RAD51C and XRCC3) have yet to be successfully purified for biochemical assays. Genetic studies have made little progress as knockout cell lines for any one of the paralogs are not viable. This proposal will utilize novel methods to overcome these obstacles and better characterize the roles of the RAD51 paralogs in HR. The first approach will determine whether specific domains mediate an interaction between RAD51 paralog proteins and BRCA2 or PALB2 (Partner and Localizer of BRCA2). Second, the RAD51 paralogs will be purified from human cells using a unique and innovative protocol we have developed. The individual paralogs, as well as the two paralog complexes, will be tested to determine if synergy exists with BRCA2 in stimulating RAD51-mediated DNA strand exchange. In a complementary cell biological approach, I will use a conditional system to systematically deplete individual RAD51 paralogs, and their respective complexes, to observe if RAD51 foci formation is compromised in response to DNA damage. Finally, in collaboration with Dr. Eli Rothenberg at NYU, we will utilize his expertise in super-resolution microscopy to test the hypothesis that the RAD51 paralog complexes are recruited to DSBs to perform specific functions during HR. Using this technique, I will create a timeline of recruitment and retention at DSBs in the context of other HR proteins. This spatiotemporal information will inform us at which step in HR the paralogs are active and their spatial relationships at a single DSB. Upon completion of the research and training fellowship, the RAD51 paralogs will be significantly more understood, and the applicant will have received extensive training. The multidisciplinary mentoring team will prepare the applicant for research independence and a successful career as a cancer researcher.