Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

M. B. Secretan, Z. Scuric, J. Oshima, A. J.R. Bishop, N. G. Howlett, D. Yau, R. H. Schiestl

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.

Original languageEnglish (US)
Pages (from-to)351-364
Number of pages14
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume554
Issue number1-2
DOIs
StatePublished - Oct 4 2004
Externally publishedYes

Keywords

  • DNA double-strand breaks
  • DSB
  • EGFP
  • EYFP
  • HR
  • MCS
  • NHEJ
  • PSS
  • double-strand break(s)
  • enhanced green fluorescent protein
  • enhanced yellow fluorescent protein
  • homologous recombination
  • multiple cloning site
  • non-homologous end-joining
  • protruding single strands

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

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