Deficient nonhomologous end-joining activity in cell-free extracts from BRCA1-null fibroblasts

Qing Zhong, Thomas G. Boyer, Phang Lang Chen, Wen Hwa Lee

Research output: Contribution to journalArticle

109 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3966-3970
Number of pages5
JournalCancer research
Volume62
Issue number14
StatePublished - Jul 15 2002

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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    Zhong, Q., Boyer, T. G., Chen, P. L., & Lee, W. H. (2002). Deficient nonhomologous end-joining activity in cell-free extracts from BRCA1-null fibroblasts. Cancer research, 62(14), 3966-3970.