Breast cancer is the most frequent malignancy affecting women in the United States. Hereditary breast and ovarian cancer represent 5-10 percent of all cases, most of which are attributed to inherited mutations in the tumor suppressor gene BRCA1. These mutations are highly penetrant and confer a 56-85 percent lifetime risk. The human BRCA1 gene encodes a protein product, which is likely to be multifunctional. The exact role of BRCA1 is still unclear but recent evidence points to the involvement of BRCA1 in two fundamental cellular processes: transcriptional regulation and DNA repair. To date, there is a wealth of evidence implicating BRCA1 in a variety of different DNA repair processes yet none of the evidence indicates a potential biological mechanism by which BRCA1 may act. These functions of BRCA1 may reflect two separate activities or, alternatively, part or all of the functions of BRCA1 in maintaining genome integrity may be an indirect result of BRCA1-mediated regulation of gene expression. Both scenarios are consistent with the current evidence. Our long-range goal is to test our central hypothesis that BRCA1 modulates transcription and that the modulation is important for efficient DNA repair. We have generated strong preliminary data for the role of BRCA1 in transcription. In addition, we have discovered a cancer-predisposing allele of BRCA1 that displays temperature-sensitive activity, providing a novel experimental system. Temperature-sensitive (TS) mutants constitute an invaluable tool to probe for the function of a particular protein. The objective of this application, which is one step towards our long-range goal, is to develop assays and characterize reagents that will be used to test whether transcriptional activation and control of DNA repair processes are interconnected functions of BRCA1. The rationale for the proposed research is that knowledge of the role of BRCA1 will allow us to design innovative approaches to counteract the effects of BRCA1 deficiency in cells and to develop functional assays for risk assessment. We will achieve our objective by accomplishing the following three specific aims: 1) Using our temperature-sensitive mutant of BRCA1, we will determine the time course of BRCA1 requirement for resistance to gamma-irradiation. 2) Establish whether subnuclear localization or hyperphosphorylation of TS BRCA1 are compromised at restrictive temperatures. 3) Determine the effects of DNA damage on transcription regulation by BRCA1. It is our expectation that the proposed research will ultimately determine the role of BRCA1 in the regulation of transcription and its connections to DNA damage response. These results will have considerable biomedical significance in identifying new targets for preventive and therapeutic intervention against women's cancers.
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