Mutations in the BRCA2 gene cause familial breast and ovarian cancers of a high penetrance, engender a strong risk to pancreatic and other cancers, and can also lead to the cancer-prone syndrome Fanconi anemia. BRCA2-deficient cells exhibit elevated chromosomal aberrations and are hypersensitive to ionizing radiation, DNA alkylating agents, and DNA crosslinking agents. Genetic studies have shown an involvement of BRCA2 in homologous recombination (HR) and the HR-mediated repair of damaged chromosomes. BRCA2 binds ssDNA and associates with the Rad51 recombinase, which catalyzes DNA joint formation in HR reactions via a filamentous polymer assembled on ssDNA. Several lines of evidence have implicated BRCA2 in the assembly of the Rad51-ssDNA nucleoprotein filament, and our preliminary studies revealing a recombination mediator activity in a BRCA2-derived polypeptide have provided compelling experimental support for this premise. To dissect the hierarchy of protein-protein and protein-ligand interactions that underlie the HR function of BRCA2, the following molecular studies will be carried out. Specifically, we will (1) define the DNA binding properties of BRCA2, (2) ascertain the functional significance of the BRCA2 DNA binding activity and of Rad51-BRCA2 interaction by characterizing variants that harbor mutations in the DNA binding domain and determining the functionality of the 8 BRC repeats of BRCA2, and (3) test hypotheses concerning influence of the BRCA2-associated protein DSS1 on the DNA binding and recombination mediator activities of BRCA2 and RPA displacement from ssDNA. Our research endeavors will help delineate the HR and tumor suppressor functions of BRCA2 and provide a paradigm for understanding the action mechanism of other recombination mediators. The experimental systems devised herein are highly suited to assessing the functional consequences of BRCA2 mutations in cancer susceptible individuals. Our studies are therefore highly germane for cancer etiology, as the molecular information derived from them can be exploited to advance cancer diagnosis, prognosis and treatment. ? ? ?
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