Nucleic Acids Research Advance Access published online on March 10, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn094
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Molecular Biology |
Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks
1Laboratory of Radiobiology & Experimental Radiation Oncology, Department of Radiotherapy and Radiation Oncology, University Medical School Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany 2Department of Tumor Biology, National Cancer Institute, University of Cairo, Egypt and 3Laboratory of Cellular & Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
*To whom correspondence should be addressed. Tel: +49 40 42803 3930; Fax: +49 40 42803 5139; Email: dahm{at}uke.uni-hamburg.de
Received January 31, 2008. Revised February 15, 2008.
Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4–/– compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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