Nucleic Acids Research Advance Access originally published online on June 6, 2008
Nucleic Acids Research 2008 36(12):4088-4098; doi:10.1093/nar/gkn347
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Nucleic Acids Research, 2008, Vol. 36, No. 12 4088-4098
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks
1Laboratory of Radiobiology & Experimental Radiation Oncology, Department of Radiotherapy and Radiation Oncology, 2Institute of Immunology, University Medical School Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany 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 (0) 40 428033930; Fax: 0049 40 42803 5139; Email: dahm{at}uke.uni-hamburg.de
Received February 5, 2008. Revised May 7, 2008. Accepted May 13, 2008.
In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51.