Genetic Dissection of BRCA1's Recombination Function

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DESCRIPTION (provided by applicant): Our goal is to understand the functions of BRCA1 that contribute to its role as a tumor suppressor gene. 80% of female mutation carriers will develop breast cancer in their lifetime. BRCA1 and the other major breast cancer susceptibility gene BRCA2 have been implicated in recombinational repair of DNA damage by promoting the assembly of the homologous recombinase RAD51 at damaged sites. BRCA1's function does not appear to be limited to promoting recombinational repair, however. A large amount of work has implicated BRCA1 in diverse biological processes suggesting that the protein is multifunctional. BRCA1 is a very large protein and interacts, directly or indirectly, with many proteins and with DNA. These different protein-protein and proten-DNA interactions have been mapped to different regions of BRCA1 suggesting a structure composed of functional modules. In spite of the large amount of work on the protein, there is relatively little known about how these different interactions relate to BRCA1's different cellular functions in recombinational repair, base-excision repair, cell cycle checkpoint control, and gene expression. Which regions of the protein contribute to a given function? How are the different functions of the functions related? Is DNA binding required for all BRCA1's functions or just a subset? Through what mechanism does BRCA1 contribute to resistance to agents that block replication fork progression? These questions are addressed using the chicken pre-B cell line DT40 because of the ease with which this line can be modified by molecular genetics. The aims of the project are as follows. 1. To analyze the phenotypes of a BRCA1-/- null mutant derivative of DT40 with respect to homologous recombination, sensitivity to DNA damage, cell cycle checkpoint control, and transcription regulation. These phenotypes will be compared to those conferred by mutation of the XRCC3 gene, which is also required for assembly of recombination complexes 2. To search for BRCA1 regulated transcripts by expression profiling and to characterize the function of a newly discovered BRCA1 regulated gene. 3. To develop a new assay for examining structural changes of stalled replication forks and determine BRCA1's role in these changes. 4. To construct and characterize a set of cell lines in which regions of BRCA1 are systematically deleted or altered to map functional domains of BRCA1 in the normal cellular context. 5. To use biochemical and genetic assays to map the regions of BRCA1 that contribute to its DNA binding activity and determine which functions of BRCA1 depend on this activity.