Loss of function of either BRCA1 or BRCA2 results in genomic instability and breast cancer predisposition, although the precise mechanism from a heterozygous mutant germline to homozygous inactivation in breast cancers is unknown. BRCA1 and to a lesser extent BRCA2 have been implicated in a wide variety of cellular processes, but a property shared by both proteins is an involvement in homologous recombination (HR). HR can be utilized for the error-free repair of DNA double-strand breaks (DSBs) or for the restart of stalled or damaged DNA replication forks. If error-free HR is defective, error-prone homologous and non-homologous repair becomes more prevalent, which may result in genomic instability. Thus, defining the roles of BRCA1 and BRCA2 in HR may be the key to understanding the cancer-prone phenotype of BRCA mutation carriers. This study has two main aims, which are related to the view that BRCA1 plays a proximal and extensive role in the cellular response to DNA damage, where it links sensing and signaling of damage to effector components. (1) We will test the hypothesis that BRCA1 controls several pathways of DSB repair, while BRCA2, which is located downstream of BRCA1, only promotes the process of error-free HR. Thus, it is predicted that the error-prone repair phenotype observed in BRCAl-deficient cells is different, and perhaps more severe, than in cells without BRCA2. We will study the outcome of error-prone repair on both the DNA sequence and the chromosomal level using novel plasmid-based assay systems. (2) BRCA1 interacts with (i) the cell-cycle checkpoint regulator Chk2, which is connected to the replication-fork-associated hMus81 protein, and with (ii) the DNA damage processing and signaling complex comprised of the Mrel 1, Rad50, and NBS1 (MRN) proteins. We will test a dual model ofBRCA1, in which BRCA1 in conjunction with Chk2 promotes homologymediated repair and replication restart, while at the same time inhibits the error-prone repair activity that is inherent to the MRN complex. These experiments may support the recent epidemiological evidence that places Chk2 and the BRCA proteins in a common breast cancer-preventing pathway. The primary goal of this study is to define the roles of BRCA1 and BRCA2 in homology-mediated DSB repair. Ultimately, the defects in HR that promote carcinogenesis by causing genetic instability may also offer a novel therapeutic target against the cancers that arise in this setting.
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