Innovative Approaches for Determining the Role of BRCA2 and BRCA1 in DNA Recombinational Repair: Examination of Genetic Instability and Possible Therapeutic Uses.

摘要

Direct evidence for the involvement of the hereditary breast cancer genes BRCA1 and BRCA2 in the repair of chromosomal double- strand breaks by homologous recombination (i.e., homology-directed repair or HDR) is provided. HDR is a precise form of repair, whereas misrepair of breaks by other mechanisms compromises genomic integrity. Key to HDR pathways in mammalian cells is the Rad51 strand-exchange protein. Since the BRCA proteins interact with Rad51, we proposed that HDR is defective in cells deficient for these proteins. In this final report we describe homologous gene-targeting experiments in Brac1 and Brca2-deficient ES cells that bring in an HDR substrate containing green fluorescent protein gene repeats. Firstly, gene targeting is reduced in both cell lines although with differential effects. Secondly, repair of an induced break in the HDR, as monitored by measuring green fluorescence within the cell, is reduced in both cell lines to a similar extent. Thirdly, Brac1 deficiency causes hypersensitivity to mitomycin C but this sensitivity is correctable. Fourthly, in wild-type cells, we see an HDR defect by interfering with Rad51 by expressing a peptide from Brca2. Defects in HDR in Brca mutants may contribute to the development of early-onset breast and ovarian cancers by destabilizing the genome.