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Molecular Basis of BRCA2-mediated Repair of Chromosome Damage
BACKGROUND: Mutations in the BRCA2 gene lead to familial breast cancer. The BRCA2-encoded product physically and functionally interacts with the Rad51 recombinase, a member of the RAD52 group of proteins that mediate homologous recombination (HR) repair of chromosome damage. Consistent with this observation, BRCA2-deficient cells show impaired HR and greatly diminished assembly of DNA damage-induced nuclear Rad51 foci. Together, available evidence points to a key role of the BRCA2 protein in HR and DNA repair via an ability of this tumor suppressor to influence the recombinase activity of Rad51. OBJECTIVE/HYPOTHESIS: BRCA2 protein binds Rad51 and the small acidic protein DSS1. BRCA2 also possesses three OB (oligonucleotide and oligosaccharide binding) folds that endow it with DNA binding ability. Ideas to be tested are that: 1. the DNA binding domain in BRCA2 that encompasses the OB folds and a Tower domain is critical for the HR function of BRCA2, 2. the BRCA2-associated protein DSS1 acts as a DNA mimic in mediating the eviction of RPA from ssDNA, in order to promote the exchange of RPA by the BRCA2-Rad51 complex. SPECIFIC AIMS: Molecular studies will test the significance of the BRCA2-ligand interactions with two specific aims. 1. The role of the OB folds in the recombination mediator function of BRCA2 will be ascertained and whether the OB2-appended Tower domain modulates DNA binding and recombination mediator functions of BRCA2 will be tested. 2. Whether the BRCA2-associated polypeptide DSS1 enhances BRCA2 recombination mediator function via attenuation of the ability of the single-strand DNA binding factor RPA to bind a DNA recombination substrate will be examined. STUDY DESIGN: Systems are available in the host laboratory for the expression and purification of Rad51, RPA, DSS1, and various BRCA2-derived polypeptides, and also to define the physical and functional interactions among these HR factors. A barrage of protein biochemistry, recombination enzymology, electron microscopy, and genetic assays will be employed to test hypotheses regarding the molecular role of BRCA2 in HR and DNA repair relevant for deciphering its function in suppressing breast cancer development. POTENTIAL OUTCOMES & BENEFITS: HR is critical for DNA damage repair, maintaining genome integrity, and preventing breast cancer. Recent evidence implicates BRCA2 in modulating the efficiency of HR via the Rad51 recombinase. As new links between dysfunctional HR and breast cancer emerge, defining mechanistic interactions between BRCA2 and Rad51 becomes imperative. By deciphering functional significances of these interactions, I aim to understand how mutations in BRCA2 lead to genome instability and breast cancer. My results may lead to advances in breast cancer prevention, diagnosis, and treatment and the experimental systems devised in these studies will be invaluable tools for assessing the functional significance of cancer-associated and unclassified BRCA2 mutations.
Though a significant proportion of familial breast cancers are caused by mutations in the BRCA2 (breast cancer susceptibility 2) gene, relatively little is known about the molecular function of the BRCA2 gene product. Importantly, recent studies have begun to elucidate the functional significance of interactions between BRCA2 and Rad51, which is a key member of the RAD52 group of proteins that repair damaged chromosomes via the high fidelity pathway of homologous recombination. I propose to combine techniques of biochemistry, electron microscopy, cytology, and genetics to elucidate the biological function of BRCA2 in the repair of chromosome damage. Results from this multi-faceted approach will shed light on the link between defective homologous recombination and breast cancer development. Moreover, the experimental systems devised will provide a valuable resource for assessing functional consequences of cancer-associated and unclassified BRCA2 mutations, for facilitating efforts of other investigators, and potentially for devising specific breast cancer treatment therapies.