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The E3 ligase RNF8 regulates KU80 removal and NHEJ repair

Nature Structural & Molecular Biology volume 19, pages 201–206 (2012)Cite this article

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Abstract

The ubiquitination cascade has a key role in the assembly of repair and signaling proteins at sites of double-strand DNA breaks. The E3 ubiquitin ligase RING finger protein 8 (RNF8) triggers the initial ubiquitination at double-strand DNA breaks, whereas sustained ubiquitination requires the downstream E3 ligase RING finger protein 168 (RNF168). It is not known whether RNF8 and RNF168 have discrete substrates and/or form different ubiquitin chains. Here we show that RNF168 acts with the ubiquitin-conjugating enzyme E2 13 (UBC13) and specifically synthesizes Lys63-linked chains, whereas RNF8 primarily forms Lys48-linked chains on chromatin, which promote substrate degradation. We also find that RNF8 regulates the abundance of the nonhomologous end-joining (NHEJ) repair protein KU80 at sites of DNA damage, and that RNF8 depletion results in prolonged retention of KU80 at damage sites and impaired nonhomologous end-joining repair. These findings reveal a distinct feature of RNF8 and indicate the involvement of the ubiquitination-mediated degradation pathway in DNA damage repair.

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Figure 1: RNF8 and RNF168 promote the accumulation of different linkage-specific ubiquitin chains at sites of DNA breaks.
Figure 2: RNF8 promotes Lys48-linked ubiquitin chain formation independent of UBC13.
Figure 3: RNF8 regulates the expression of KU80 and CHK2 in vivo.
Figure 4: Accumulation of Lys48-linked and Lys63-linked ubiquitin chains at DNA damage sites.

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References

  1. Weissman, A.M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell Biol. 2, 169–178 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Komander, D. et al. Molecular discrimination of structurally equivalent Lys63–linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Eddins, M.J., Varadan, R., Fushman, D., Pickart, C.M. & Wolberger, C. Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH. J. Mol. Biol. 367, 204–211 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243, 1576–1583 (1989).

    Article  CAS  PubMed  Google Scholar 

  5. Matsumoto, M.L. et al. K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody. Mol. Cell 39, 477–484 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Raiborg, C. & Stenmark, H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458, 445–452 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Kirkin, V., McEwan, D.G., Novak, I. & Dikic, I. A role for ubiquitin in selective autophagy. Mol. Cell 34, 259–269 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Hofmann, R.M. & Pickart, C.M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Wu-Baer, F., Lagrazon, K., Yuan, W. & Baer, R. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin. J. Biol. Chem. 278, 34743–34746 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Nishikawa, H. et al. Mass spectrometric and mutational analyses reveal Lys-6–linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase. J. Biol. Chem. 279, 3916–3924 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Zhao, G.Y. et al. A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol. Cell 25, 663–675 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Huen, M.S. et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 131, 901–914 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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  PubMed  Google Scholar 

  16. 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  PubMed  Google Scholar 

  17. 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  PubMed  Google Scholar 

  18. Feng, L., Wang, J. & Chen, J. The Lys63-specific deubiquitinating enzyme BRCC36 is regulated by two scaffold proteins localizing in different subcellular compartments. J. Biol. Chem. 285, 30982–30988 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Fujimuro, M., Sawada, H. & Yokosawa, H. Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins. FEBS Lett. 349, 173–180 (1994).

    Article  CAS  PubMed  Google Scholar 

  22. Kim, H., Chen, J. & Yu, X. Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response. Science 316, 1202–1205 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Wang, B. et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Plans, V. et al. The RING finger protein RNF8 recruits UBC13 for lysine 63–based self polyubiquitylation. J. Cell. Biochem. 97, 572–582 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Lorick, K.L. et al. RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA 96, 11364–11369 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fang, S., Jensen, J.P., Ludwig, R.L., Vousden, K.H. & Weissman, A.M. Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275, 8945–8951 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Postow, L. et al. Ku80 removal from DNA through double strand break–induced ubiquitylation. J. Cell Biol. 182, 467–479 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Burma, S., Chen, B.P. & Chen, D.J. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst.) 5, 1042–1048 (2006).

    Article  CAS  Google Scholar 

  29. Kim, J.S. et al. Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells. J. Cell Biol. 170, 341–347 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mari, P.O. et al. Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4. Proc. Natl. Acad. Sci. USA 103, 18597–18602 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Galanty, Y. et al. Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. Nature 462, 935–939 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li, L. et al. Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer. J. Exp. Med. 207, 983–997 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shao, G. et al. MERIT40 controls BRCA1-Rap80 complex integrity and recruitment to DNA double-strand breaks. Genes Dev. 23, 740–754 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Feng, L., Huang, J. & Chen, J. MERIT40 facilitates BRCA1 localization and DNA damage repair. Genes Dev. 23, 719–728 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang, B., Hurov, K., Hofmann, K. & Elledge, S.J. NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control. Genes Dev. 23, 729–739 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Penengo, L. et al. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin. Cell 124, 1183–1195 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Lukas, C., Falck, J., Bartkova, J., Bartek, J. & Lukas, J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5, 255–260 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Walker, J.R., Corpina, R.A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Antoni, L., Sodha, N., Collins, I. & Garrett, M.D. CHK2 kinase: cancer susceptibility and cancer therapy—two sides of the same coin? Nat. Rev. Cancer 7, 925–936 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Lok, G.T. et al. Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation. Nucleic Acids Res. 40, 196–205 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Bekker-Jensen, S. et al. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nat. Cell Biol. 12, 80–6; (suppl. pp 1–12).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank our colleagues in the Chen laboratory for insightful discussions and technical assistance. We also thank X. Zhang and H.P. Adams for technical assistance with the laser microirradiation. This work was supported in part by grants from the US National Institutes of Health (CA089239 and CA092312 to J.C.). J.C. is a recipient of an Era of Hope Scholar award from the US Department of Defense (W81XWH-05-1-0470) and is a member of the MD Anderson Cancer Center (CA016672).

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  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Lin Feng & Junjie Chen

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  1. Lin Feng
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L.F. designed and carried out the experiments. J.C. advised on the design of the experiments. L.F. and J.C. were responsible for the preparation of the manuscript.

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Correspondence to Junjie Chen.

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Feng, L., Chen, J. The E3 ligase RNF8 regulates KU80 removal and NHEJ repair. Nat Struct Mol Biol 19, 201–206 (2012). https://doi.org/10.1038/nsmb.2211

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