Role of the Breast Cancer Susceptibility 2 BRC Repeats in Homologous Recombination

• Main Author: Cealic, Iulia
• Other Authors: Baker, Mark
• Language: en
• Published: 2012
• Subjects: BRCA2; BRC repeats; Homologous recombination; Gene targeting; Rad51
• Online Access: http://hdl.handle.net/10214/5259

Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of genomic DNA. The product of the Breast Cancer Susceptibility 2 (BRCA2) gene functions as a recombination mediator in HR-directed repair of DSBs. BRCA2 interacts directly with RAD51, the central recombinase of HR, through highly conserved repetitive motifs of 30-40 amino acids, named BRC repeats, and regulates the formation of the RAD51-ssDNA nucleoprotein filament. There is significant variability in the number of BRC repeats among taxa. However, all mammalian BRCA2 orthologs have eight BRC repeats, which display different characteristics in in vitro studies of RAD51-ssDNA nucleoprotein filament. To test the importance of the number of BRC repeats and to evaluate the role of individual BRC repeats in HR, BRCA2 variants bearing different combinations of BRC repeats were generated using BAC-recombineering, expressed in murine hybridoma cells, and assayed for the ability to stimulate HR using a gene targeting assay. The BRCA2 variant bearing BRC repeats 1 to 4 decreased the efficiency of HR and increased the level of Rad51 protein, whereas the BRCA2 variant bearing BRC repeats 5 to 8 significantly stimulated HR, but had no effect on the level of Rad51. These results supported the hypothesis that BRC repeats are not functionally equivalent, but rather have different, perhaps reinforcing functions in HR. === Canadian Institutes of Health Research