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The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

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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Figure 1: The p97–UFD1–NPL4 ATPase complex is involved in the response to DNA DSBs.
Figure 2: p97–UFD1–NPL4 is essential for DSB repair and survival after exposure to ionizing radiation.
Figure 3: p97 is recruited to DSB sites in an RNF8-dependent manner.
Figure 4: p97 turns over RNF8-dependent K48–Ub conjugates at damage sites.
Figure 5: p97–UFD1–NPL4 governs BRCA1, 53BP1 and RAD51 localization to DSB sites.

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References

  1. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  Google Scholar 

  2. Kinner, A., Wu, W., Staudt, C. & Iliakis, G. γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res. 36, 5678–5694 (2008).

    Article  CAS  Google Scholar 

  3. van Attikum, H. & Gasser, S. M. Crosstalk between histone modifications during the DNA damage response. Trends Cell Biol. 19, 207–217 (2009).

    Article  CAS  Google Scholar 

  4. Bergink, S. & Jentsch, S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458, 461–467 (2009).

    Article  CAS  Google Scholar 

  5. Panier, S. & Durocher, D. Regulatory ubiquitylation in response to DNA double-strand breaks. DNA Repair (Amst) 8, 436–443 (2009).

    Article  CAS  Google Scholar 

  6. Ulrich, H. D. & Walden, H. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489 (2010).

    Article  CAS  Google Scholar 

  7. Al-Hakim, A. et al. The ubiquitous role of ubiquitin in the DNA damage response. DNA Repair (Amst) 9, 1229–1240 (2010).

    Article  CAS  Google Scholar 

  8. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

    Article  CAS  Google Scholar 

  9. Doil, C. et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136, 435–446 (2009).

    Article  CAS  Google Scholar 

  10. Stewart, G. S. et al. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136, 420–434 (2009).

    Article  CAS  Google Scholar 

  11. Meusser, B., Hirsch, C., Jarosch, E. & Sommer, T. ERAD: the long road to destruction. Nat. Cell Biol. 7, 766–772 (2005).

    Article  CAS  Google Scholar 

  12. Jentsch, S. & Rumpf, S. Cdc48 (p97): a ‘molecular gearbox’ in the ubiquitin pathway? Trends Biochem. Sci. 32, 6–11 (2007).

    Article  CAS  Google Scholar 

  13. Ye, Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase. J. Struct. Biol. 156, 29–40 (2006).

    Article  CAS  Google Scholar 

  14. Yeung, H. O. et al. Insights into adaptor binding to the AAA protein p97. Biochem. Soc. Trans. 36, 62–67 (2008).

    Article  CAS  Google Scholar 

  15. Richly, H. et al. A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 120, 73–84 (2005).

    Article  CAS  Google Scholar 

  16. Alexandru, G. et al. UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1α turnover. Cell 134, 804–816 (2008).

    Article  CAS  Google Scholar 

  17. Meyer, H. & Popp, O. Role(s) of Cdc48/p97 in mitosis. Biochem. Soc. Trans. 36, 126–130 (2008).

    Article  CAS  Google Scholar 

  18. Rape, M. et al. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone. Cell 107, 667–677 (2001).

    Article  CAS  Google Scholar 

  19. Ye, Y., Meyer, H. H. & Rapoport, T. A. Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J. Cell Biol. 162, 71–84 (2003).

    Article  CAS  Google Scholar 

  20. Ramadan, K. et al. Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin. Nature 450, 1258–1262 (2007).

    Article  CAS  Google Scholar 

  21. Verma, R., Oania, R., Fang, R., Smith, G. T. & Deshaies, R. J. Cdc48/p97 mediates UV-dependent turnover of RNA Pol II. Mol. Cell 41, 82–92 (2011).

    Article  CAS  Google Scholar 

  22. Livingstone, M. et al. Valosin-containing protein phosphorylation at Ser784 in response to DNA damage. Cancer Res. 65, 7533–7540 (2005).

    Article  CAS  Google Scholar 

  23. Mouysset, J. et al. Cell cycle progression requires the CDC-48UFD-1/NPL-4 complex for efficient DNA replication. Proc. Natl Acad. Sci. USA 105, 12879–12884 (2008).

    Article  CAS  Google Scholar 

  24. Zhang, H., Wang, Q., Kajino, K. & Greene, M. I. VCP, a weak ATPase involved in multiple cellular events, interacts physically with BRCA1 in the nucleus of living cells. DNA Cell Biol. 19, 253–263 (2000).

    Article  CAS  Google Scholar 

  25. Potts, P. R. & Yu, H. Human MMS21/NSE2 is a SUMO ligase required for DNA repair. Mol. Cell. Biol. 25, 7021–7032 (2005).

    Article  CAS  Google Scholar 

  26. Morris, J. R. et al. The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462, 886–890 (2009).

    Article  CAS  Google Scholar 

  27. Claessen, J. H., Mueller, B., Spooner, E., Pivorunas, V. L. & Ploegh, H. L. The transmembrane segment of a tail-anchored protein determines its degradative fate through dislocation from the endoplasmic reticulum. J. Biol. Chem. 285, 20732–20739 (2010).

    Article  CAS  Google Scholar 

  28. Bekker-Jensen, S. et al. Spatial organization of the mammalian genomesurveillance machinery in response to DNA strand breaks. J. Cell Biol. 173, 195–206 (2006).

    Article  CAS  Google Scholar 

  29. O’Driscoll, M., Ruiz-Perez, V. L., Woods, C. G., Jeggo, P. A. & Goodship, J. A. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet. 33, 497–501 (2003).

    Article  Google Scholar 

  30. Matsumoto, M. et al. Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells. J. Cell Sci. 120, 1104–1112 (2007).

    Article  CAS  Google Scholar 

  31. Hanada, K. et al. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat. Struct. Mol. Biol. 14, 1096–1104 (2007).

    Article  CAS  Google Scholar 

  32. Kolas, N. K. et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318, 1637–1640 (2007).

    Article  CAS  Google Scholar 

  33. Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007).

    Article  CAS  Google Scholar 

  34. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008).

    Article  CAS  Google Scholar 

  35. Ramadan, K. & Meerang, M. Degradation-linked ubiquitin signal and proteasome are integral components of DNA double strand break repair: New perspectives for anti-cancer therapy. FEBS Lett. 585, 2868–2875 (2011).

    Article  CAS  Google Scholar 

  36. Sy, S. M. et al. Critical roles of ring finger protein RNF8 in replication stress responses. J. Biol. Chem. 286, 22355–22361 (2011).

    Article  CAS  Google Scholar 

  37. Stewart, G. S. et al. RIDDLE immunodeficiency syndrome is linked to defects in 53BP1-mediated DNA damage signaling. Proc. Natl Acad. Sci. USA 104, 16910–16915 (2007).

    Article  CAS  Google Scholar 

  38. Kirkin, V. & Dikic, I. Ubiquitin networks in cancer. Curr. Opin. Genet. Dev. 21, 21–28 (2011).

    Article  CAS  Google Scholar 

  39. Ju, J. S., Miller, S. E., Hanson, P. I. & Weihl, C. C. Impaired protein aggregate handling and clearance underlie the pathogenesis of p97/VCP-associated disease. J. Biol. Chem. 283, 30289–30299 (2008).

    Article  CAS  Google Scholar 

  40. Ritz, D. et al. Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations. Nat. Cell Biol. 13, 1116–1123 (2011).

    Article  CAS  Google Scholar 

  41. Wojcik, C., Yano, M. & DeMartino, G. N. RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J. Cell Sci. 117, 281–292 (2004).

    Article  CAS  Google Scholar 

  42. Hosokawa, N. et al. Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP. J. Biol. Chem. 283, 20914–20924 (2008).

    Article  CAS  Google Scholar 

  43. Goldberg, M. et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956 (2003).

    Article  CAS  Google Scholar 

  44. Ahnesorg, P., Smith, P. & Jackson, S. P. XLF interacts with the XRCC4-DNA ligaseIV complex to promote DNA nonhomologous end-joining. Cell 124, 301–313 (2006).

    Article  CAS  Google Scholar 

  45. Kratz, K. et al. Deficiency of FANCD2-associated nuclease KIAA1018/FAN1 sensitizes cells to interstrand crosslinking agents. Cell 142, 77–88 (2010).

    Article  CAS  Google Scholar 

  46. Bennardo, N., Cheng, A., Huang, N. & Stark, J. M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008).

    Article  Google Scholar 

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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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Correspondence to Hemmo Meyer or Kristijan Ramadan.

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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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