Roles of BRCA2 Gene in Homologous Recombination and Genomic Stability
Abstract
Primary and spontaneously immortalized mouse embryonic fibroblasts (MEF) were derived from embryonal stem cells in which both alleles of the BRCA2 gene have been mutated by gene targeting. The mutation deletes an extreme carboxy-terminal domain of the BRCA2 protein encoded by exon 27, one of two regions known to mediate binding with RAD51 protein. BRCA2-mutant MEF were markedly hypersensitive to the DNA crosslinking drug mitomycin-C, and showed evidence of severe chromosomal instability. While immortal wild-type MEF approximated tetraploidy, immortal BRCA2-mutant MEF showed subtetraploid chromosome numbers and one to several abnormally small chromosomes or "minichromosomes" in every metaphase. The majority of minichromosomes either lacked telomeres or lacked a centromere detectable by fluorescence in-situ hybridization. Primary MEF were then examined for chromosomal changes over the first few passages after dissociation from embryos. BRCA2-mutant primary MEF showed sharply elevated numbers of chromatid and chromosome aberrations, including breaks, deletions and exchanges. By the third passage, approximately half the metaphase cells in BRCA2-mutant populations had one or more chromosome abnormalities. This marked chromosomal instability in BRCA2-mutant cells, and their hypersensitivity to DNA interstrand crosslinks, may reflect a deficiency in DNA repair by homologous recombination.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2000
- Accession Number
- ADA387755
Entities
People
- David Chen
Organizations
- University of California, Berkeley