Mutagenesis Advance Access originally published online on March 7, 2007
Mutagenesis 2007 22(3):217-233; doi:10.1093/mutage/gem007
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The mutagenic potential of non-homologous end joining in the absence of the NHEJ core factors Ku70/80, DNA-PKcs and XRCC4-LigIV
1Department of Biology and Geography, Institute of Genetics, University of Duisburg-Essen, Universitätsstrasse 5, D-45117 Essen, Germany 2Present address: Department of Pediatric Hematology/Oncology, University Children's Hospital of Essen, Hufelandstrasse 55, D-45122 Essen, Germany 3Present address: Institute of Biochemistry and Molecular Biology 2, University of Düsseldorf Medical School, Universitätsstrasse 1, D-40225 Düsseldorf, Germany 4Present address: Dr Fooke Laboratories GmbH, Mainstrasse 85, D-41469 Neuss, Germany 5Present address: Institute for Genetics, University of Cologne, Zülpicherstrasse 47, D-50674 Köln, Germany
Non-homologous end joining (NHEJ), the major pathway of double-strand break (DSB) repair in mammalian cells, comprises two subpathways: one that requires the three core factors Ku70/80, DNA-PKcs and XRCC4/LigIV (DNA-PK-dependent NHEJ) and the other that is independent of these factors. Using a cell-free NHEJ assay, we have investigated the ability of three Chinese hamster ovary (CHO) mutants deficient in Ku80 (xrs6), DNA-PKcs (XR-C1) and XRCC4 (XR-1) in comparison with CHO-K1 wild-type cells to rejoin non-compatible DSB ends. Both NHEJ efficiency and fidelity are strongly reduced in the mutants with xrs6 and XR-1 exhibiting the strongest reduction and XR-C1 displaying a phenotype intermediate between the wild-type and the other two mutants indicating a non-essential but facilitating role of DNA-PKcs in NHEJ. The decrease in fidelity in the mutants is expressed by an increase of deletion junctions formed at microhomologies (µhom) near the DSB (microhomology-mediated non-homologous end joining: µhomNHEJ). Using a novel µhomNHEJ assay, we show that µhom regions of 6–10 bp that are located directly at the DSB termini strongly enhance the mutagenic µhomNHEJ reaction even in the wild type. Due to its error proneness, DNA-PK-independent µhomNHEJ may actively promote genome instability. It will, therefore, be of increasing importance to examine NHEJ fidelity in the context with tumorigenesis and cellular senescence for which we here provide two efficient and reliable tools.
* To whom correspondence should be addressed. Email: petra.pfeiffer{at}uni-koeln.de
Received on November 11, 2006; revised on January 18, 2007; accepted on January 22, 2007.
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