Deficient nonhomologous end-joining activity in cell-free extracts from Brca1-null fibroblasts.
BRCA1 ensures genomic stability, at least in part, through a functional role in DNA damage repair. BRCA1 interacts with the Rad50/Mre11/Nbs1 complex that occupies a central role in DNA double-strand break repair mediated by homologous recombination and nonhomologous end joining (NHEJ). NHEJ can be catalyzed by mammalian whole cell extract in a reaction dependent upon DNA ligase IV, Xrcc4, Ku70, Ku80, and DNA-PKcs. Here, we show that under identical cell-free reaction conditions, the addition of antibodies specific for BRCA1 and Rad 50 but not Rad51, inhibits end-joining activity. Cell extracts derived from Brca1-deficient mouse embryonic fibroblasts exhibit reduced end-joining activity independent of the endogenous protein amounts of DNA ligase IV, Ku80, and Ku70. The Brca1-dependent NHEJ activity predominates at the lower concentrations of Mg2+ (0.5 mM); elevated Mg2+ or Mn2+ concentrations (10 mM) dramatically increase overall end-joining activity and abrogates the requirement for Brca1, Xrcc4, and Ku70. The addition of partially purified BRCA1, in association with Rad50/Mre11/Nbs1 complex, complements the NHEJ deficiency of Brca1-null fibroblast extracts. These results suggest a role for Brca1 in NHEJ and in the maintenance of genome integrity.
Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
Qing
Q
Zhong
Thomas G
TG
Boyer
Phang-Lang
PL
Chen
Wen-Hwa
WH
Lee
D000818:Animals; D019313:BRCA1 Protein / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D002457:Cell Extracts / Q000494:pharmacology; D002460:Cell Line; D002474:Cell-Free System; D004260:DNA Repair / Q000502:physiology*; D005347:Fibroblasts / Q000737:chemistry / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000502:physiology; D051379:Mice
D019313:BRCA1 Protein; D002457:Cell Extracts