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 language | English (US) |
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Pages (from-to) | 351-364 |
Number of pages | 14 |
Journal | Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis |
Volume | 554 |
Issue number | 1-2 |
DOIs | |
State | Published - Oct 4 2004 |
Externally published | Yes |
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