Abstract
Here we have used an intrachromosomal substrate to monitor the end joining of distant ends, which leads to DNA rearrangements in mammalian cells. We show that silencing Mre11 reduces the efficiency of nonhomologous end joining (NHEJ), affecting both the canonical and alternative pathways, partly in a manner that is independent of the ataxia-telangiectasia mutated kinase (ATM). Silencing of Rad50 or CtIP decreases end-joining efficiency in the same pathway as Mre11. In cells defective for Xrcc4, the MRE11–RAD50–NBS1 (MRN) complex inhibitor MIRIN decreases end-joining frequencies, demonstrating a role for MRN in alternative NHEJ. Consistently, MIRIN sensitizes both complemented and NHEJ-defective cells to ionizing radiation. Conversely, overexpression of Mre11 stimulates the resection of single-stranded DNA and increases alternative end joining, through a mechanism that requires Mre11's nuclease activity, but in an ATM-independent manner. These data demonstrate that, in addition to its role in ATM activation, Mre11 can favor alternative NHEJ through its nuclease activity.
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Acknowledgements
We thank Y. Shiloh (Sackler School of Medecine, Tel Aviv University) for providing the wild-type and mutant MRE11 plasmids, M. Jasin (Memorial Sloan-Kettering Cancer Center) for donating the I-SceI expression vector and R. Baer (Columbia University Medical Center) for CtIP antibodies. We are grateful to R. Scully (Beth Israel Deaconess Medical Center) and D. Ferguson (University of Michigan Medical School) for sharing results and to S. Gangloff, V. Pennaneach (IRCM), Y. Canitrot and D. Trouche (Université Paul Sabatier) for helpful discussions. We apologize to authors that are not quoted in references, due to space limitations. Thanks to G. Palierne for efficient technical support. E.R. is supported by a fellowship from La Ligue Nationale contre le Cancer and A.G. is an IRTELIS laureate. This work was supported by grants from La Ligue Nationale contre le Cancer “Equipe labellisée LA LIGUE 2008”, Agence Nationale de la Recherche and Institut National du Cancer.
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E.R. and A.G. performed experiments, participated in the interpretations and in the manuscript preparation; I.P. started the first experiments; J.G. furnished the MIRIN and the conditions of use; P.B. conceived the experiments, participated in the experiments and in the manuscript preparation and interpretated the data; B.S.L. conceived and interpreted the experiments and wrote the manuscript.
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Rass, E., Grabarz, A., Plo, I. et al. Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells. Nat Struct Mol Biol 16, 819–824 (2009). https://doi.org/10.1038/nsmb.1641
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DOI: https://doi.org/10.1038/nsmb.1641
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