) BRCA1 and BRCA2, suppressors of breast cancer formation, have been proposed to be involved in DNA repair. We showed that BRCA2 interacts directly with Rad51 and these interactions, mediated by conserved BRC repeats, are necessary for the ability of BRCA2 to restore the resistance of BRCA2-deficient cells to DNA-damage agents. Recent results also showed that BRCA1 is important in DNA repair, probably acting through the Rad50/MRE11/p95 complex. To elucidate the biological significance of the interactions of BRCA1 and BRCA2 with proteins involved in the DNA repair machinery, the following specific aims are proposed:
Aim 1 is to elucidate the functional importance of the interaction between Rad51 and BRC repeats in BRCA2 for DNA DSB repair by three steps. The first step is to conditionally overexpress fusions of the GFP and wild-type or mutated BRC repeats for testing the effects on cell survival, cell cycle checkpoint, apoptosis and chromosome stability in response to DNA damage. Second is to specifically test whether the BRC/Rad51 interaction is crucial for such activity by expressing BRCA2 with single point mutations in the BRC repeats in mouse Brca2-/-; p53-/- fibroblasts. Mutant BRCA2 will be compared with the wild-type for their effects in response to DNA damage. The third is to test whether BRCA2 influences the biochemical activities of Rad51.
Aim 2 is to investigate the role of BRCA1 in the DNA repair process by testing the formation of the complex containing BRCA1, Mre11/Rad50/p95, and RLB1 or MLB1. The signal transduction pathway(s) leading to the formation of this complex are also to be examined.
Aim 3 is to establish the roles of two novel proteins, MLB1 or RLB1, in DNA repair by generating mice with their genes inactivated by homologous recombination. These mice will be examined for the incidence of tumor formation and for their relative sensitivity to ionizing radiation. In addition, mice with mammary gland-specific knockout using the Cre lox system combined with the binary tetracycline gene control system will be tested for their sensitivity to ionizing radiation. Substantiating a link between BRCA1/2 with DNA repair will have a major impact on clinical applications. Compounds that specifically interrupt the BRCA2/Rad51 or BRCA1/Rad50 complexes can be developed to render targeted tumor cells hypersensitive to radiation or chemotherapy drugs. Tumor cells could then be killed with lower doses thereby reducing the deleterious side effects of treatment.
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