Abstract
Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death1. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion2,3. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood4,5,6,7. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1–NPL4 and Lys-48-linked ubiquitin (K48–Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48–Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms3,7,8. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97–UFD1–NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.
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Acknowledgements
We thank C. C. Weihl (Department of Neurology, Washington University School of Medicine, Saint Louis, Missouri, USA) for providing stable transfected U2OS cell lines, J. Stark (Department of Radiation Biology, Beckman Research Institute of City of Hope, Duarte, California, USA) for EJ5–GFP and DR–GFP HEK293 cell lines, J. Lukas (Centre for Genotoxic Stress Research, Institute of Cancer Biology and Danish Cancer Society, Strandboulevarden, Copenhagen, Denmark) for the RNF8–Flag construct, L. Penengo (Laboratory of Molecular Biology, DiSCAFF Department, Novara, Italy) for the RNF168–Flag construct, H. Ploegh (Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA) for the SEL1L antibody and Genentech for the K63–Ub and K48–Ub antibodies. We thank T. Ramadan, H. Nägeli, M. Stucki and A. Sartori for critical reading of the manuscript, M. Lopes and K. Neelsen for help with establishing the mPFGE method, B. Mihaljevic for help with establishing the laser micro-irradiation method and M. Vitanescu for technical assistance. This work was supported by the ‘Forschungskredit’ (55100203) of the University of Zürich to K.R., Novartis Stiftung für Biologisch-Medizinische Forschung (09B48) to K.R. for M.M., the Swiss National Research Foundation to H.M. and P.J. and an Oncosuisse grant to U.H. This project was initiated at the Institute of Veterinary Biochemistry and Molecular Biology by K.R. We also thank the Center for Microscopy and Image Analysis of the University of Zürich-Irchel, for providing the confocal microscope and their assistance with image analysis.
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M.M. carried out most of the experiments in the manuscript. D.R. created inducible stable HEK293 cell lines expressing wild-type p97 or p97EQ. S.P. carried out NHEJ and homologous recombination reporter assays. Z.G. carried out the K48–Ub immunofluorescence microscopy study. M.B. carried out FACS analysis. N.M. provided the stable transfected RNF8-shRNA-expressing U2OS cell line. K.R. initiated the project, carried out mPFGE experiments and conceived the study. K.R. and H.M. designed the experiments and wrote the manuscript. All authors discussed the experiments and gave suggestions for the manuscript.
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Meerang, M., Ritz, D., Paliwal, S. et al. The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks. Nat Cell Biol 13, 1376–1382 (2011). https://doi.org/10.1038/ncb2367
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DOI: https://doi.org/10.1038/ncb2367
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