Investigating contributions of BRCA2 DNA-binding domains toward maintaining genome integrity

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ABSTRACT Conserved mechanisms exist to eliminate DNA damage, which, if left unrepaired, can cause gene mutations and genome rearrangements to trigger neoplastic cell transformation and oncogenesis. In particular, homologous recombination (HR) represents a high-fidelity tool for the repair of DNA double-strand breaks (DSBs), interstrand crosslinks, and collapsed DNA replication forks. Proper execution of HR requires the tumor suppressor BRCA2 (Breast Cancer 2). Germline mutations in BRCA2 lead to breast and ovarian cancer and also the cancer-prone syndrome Fanconi anemia. Moreover, somatic driver BRCA2 mutations are found in a variety of cancer types. Further work is necessary to elucidate the mechanistic role of BRCA2 in HR repair. Importantly, the research proposed herein will help define the contributions of two distinct DNA binding domains toward the function of BRCA2 as a “recombination mediator,” specifically in facilitating the assembly of complexes of the RAD51 recombinase on single-stranded DNA derived from the processing of DSBs. Our central hypothesis posits that the two DNA-binding domains within BRCA2 play an essential role in guiding BRCA2-dependent DSB repair and replication fork preservation through the engagement of single-stranded DNA and DNA structures present in processed DSBs. We will conduct a variety of biochemical, genetic, and cell biological studies under two specific aims to test this central tenet. Throughout these experiments, we will delineate how genetic mutations within each DNA binding domain affect key aspects of HR and replication fork preservation. The results from this fellowship project will not only expand our understanding of how the BRCA2 DNA-binding domains contribute to the maintenance of genome stability, but they will also shed light on how mutations within these domains ultimately cause malignancy. Furthermore, we expect our findings to help identify new targets and pathway pivot points for the development of novel cancer therapeutics.