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(PP328) The role of BRCA2 in Rad51 localization during breast carcinogenesis.
Malone, Jennifer*,, Lieberman, Rita, Holt, Jeff,
ABSTRACT- Objective/Hypothesis: BRCA2 binds Rad51 and facilitates its role in DNA double strand break repair (DDSB). Our laboratory and others have illustrated that Rad51 is mislocalized to the cytoplasm in BRCA2 hereditary breast cancer cells. Since Rad51 is known to bind to the BRC repeats and potentially the C-terminal domain of BRCA2, it is postulated that BRCA2 may function in the nuclear translocation of Rad51 since it lacks its own nuclear localization signal (NLS). Our hypothesis is that the mislocalization of Rad51 induces breast carcinogenesis due to alterations in the phosphorylation of BRCA2. Specific Aims: The specific aims of this study are: 1. Determine Rad51 localization during breast cancer progression; 2. Determine Rad51 function in the DNA damage response in sporadic breast cancers; 3. Determine if the mislocalization of Rad51 leads to breast carcinogenesis; 4. Determine if BRCA2 phosphorylation has a role in Rad51 function in DNA repair. Methods: To determine if the mislocalization of Rad51 results in defective DNA repair, Rad51 mutants which mislocalize to the cytoplasm will be engineered by site-directed mutagenesis. Site-directed mutagenesis will also be targeted to regions in BRCA2 where Rad51 binds in order to determine if the phosphorylation status affects the localization and binding of Rad51. Study Design: Rad51 mutants with an added NLS will be tested to verify if they can reverse mutant BRCA2 malignancy in CHO cells. Cells will be transfected with the Rad51 mutants with and without Rad51-targeted siRNA to verify if the transformation is due to the mislocalized Rad51 being ineffective at DNA damage repair. The BRCA2 mutants will be co-transfected along with full-length BRCA2 expression vectors in CAPAN-1 cells in order to analyze Rad51 binding at specific phosphorylation sites. This will allow for the determination of BRCA2′s involvement in Rad51 nuclear transport. Rad51 localization, radiation sensitivity, Rad51 foci formation, DDSB repair and homologous recombination will be analyzed to validate findings. Conclusions: These studies will help to determine whether the mislocalization of Rad51 contributes to the development of sporadic breast cancer. This study will also help to determine what BRCA2′s role is in the nuclear transport of Rad51 and how they interact in DDSB repair.
Key words: brca2, rad51, breast carcinogenesis
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