title: BRCA1 requirement for the fidelity of plasmid DNA double-strand break repair in cultured breast epithelial cells.
abstract: The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.
authors.affiliation: Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721, USA. ericgt@email.arizona.edu
authors.forename: Eric G
authors.initials: EG
authors.lastname: Thompson
authors.forename: Hanna
authors.initials: H
authors.lastname: Fares
authors.forename: Kathleen
authors.initials: K
authors.lastname: Dixon
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D001940:Breast / Q000166:cytology*; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids / Q000235:genetics*
chemical_list: D019313:BRCA1 Protein; D004247:DNA