BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks.
BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.
Zhong
Qing
Q
Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
Chen
Chi-Fen
CF
Chen
Phang-Lang
PL
Lee
Wen-Hwa
WH
D000256:Adenoviridae / Q000235:genetics; D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D002875:Chromosomes / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism; D019398:Genes, BRCA1; D005838:Genotype; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D004247:DNA; D008156:Luciferases