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Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass

Abstract

Protein ubiquitylation has emerged as a key regulatory mechanism in DNA-damage signalling and repair pathways. We report a proteome-wide, site-specific survey of ubiquitylation changes after ultraviolet irradiation, identifying numerous upregulated and downregulated ubiquitylation sites on known components of DNA-damage signalling, as well as on proteins not previously implicated in this process. Our results uncover a critical role for PCNA-associated factor PAF15 (p15(PAF)/KIAA0101) ubiquitylation during DNA replication. During unperturbed S phase, chromatin-associated PAF15 is modified by double mono-ubiquitylation of Lys 15 and 24 templated through PCNA binding. Replication blocks trigger rapid, proteasome-dependent removal of Lys 15/24-ubiquitylated PAF15 from PCNA, facilitating bypass of replication-fork-blocking lesions by allowing recruitment of translesion DNA synthesis polymerase polη to mono-ubiquitylated PCNA at stalled replisomes. Our findings demonstrate widespread involvement of ubiquitin signalling in genotoxic-stress responses and identify a critical function for dynamic PAF15 ubiquitylation in safeguarding genome integrity when DNA replication is challenged.

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Figure 1: Proteome-wide quantification of DNA-damage-regulated ubiquitylation sites.
Figure 2: Functional modules of proteins modified by ultraviolet-regulated ubiquitylation.
Figure 3: Rapid depletion of double mono-ubiquitylation of PAF15 at Lys 15 and 24 in response to ultraviolet irradiation.
Figure 4: Ubiquitylation of PAF15 at Lys 15 and 24 depends on the integrity of the ongoing DNA replication and its binding to PCNA.
Figure 6: Ubiquitylation of PAF15 at Lys 15 and 24 promotes polymerase switching during TLS.
Figure 5: PAF15 promotes cell survival and restart of stalled forks following replication blocks in a manner dependent on the integrity of Lys 15 and 24.
Figure 7: Model of the role of ubiquitin-dependent regulation of PAF15 in genome stability maintenance and during the cell cycle.

References

  1. Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Deshaies, R. J. & Joazeiro, C. A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    CAS  PubMed  Google Scholar 

  3. Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Ciccia, A. & Elledge, S. J. The DNA damage response: Making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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 

  7. 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 

  8. Bekker-Jensen, S. & Mailand, N. Assembly and function of DNA double-strand break repair foci in mammalian cells. DNA Repair (Amst) 9, 1219–1228 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Lehmann, A. R. et al. Translesion synthesis: Y-family polymerases and the polymerase switch. DNA Repair (Amst) 6, 891–899 (2007).

    Article  CAS  Google Scholar 

  11. Moldovan, G. L., Pfander, B. & Jentsch, S. PCNA, the maestro of the replication fork. Cell 129, 665–679 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Friedberg, E. C. Suffering in silence: the tolerance of DNA damage. Nat. Rev. Mol. Cell Biol. 6, 943–953 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Bienko, M. et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, 1821–1824 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Huang, T. T. et al. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat. Cell Biol. 8, 339–347 (2006).

    CAS  PubMed  Google Scholar 

  15. Wagner, S. A. et al. A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol. Cell Proteomics 10, M111 013284 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Emanuele, M. J. et al. Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xu, G., Paige, J. S. & Jaffrey, S. R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gillet, L. C. & Scharer, O. D. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem. Rev. 106, 253–276 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Sugasawa, K. et al. UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex. Cell 121, 387–400 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Xiao, A. et al. WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity. Nature 457, 57–62 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401–1412 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Emanuele, M. J., Ciccia, A., Elia, A. E. & Elledge, S. J. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate. Proc. Natl Acad. Sci. USA 108, 9845–9850 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Williamson, A. et al. Regulation of ubiquitin chain initiation to control the timing of substrate degradation. Mol. Cell 42, 744–757 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Abbas, T. & Dutta, A. CRL4Cdt2: master coordinator of cell cycle progression and genome stability. Cell Cycle 10, 241–249 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jackson, D. A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Niimi, A. et al. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells. Proc. Natl Acad. Sci. USA 105, 16125–16130 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Choi, J. H. & Pfeifer, G. P. The role of DNA polymerase eta in UV mutational spectra. DNA Repair (Amst) 4, 211–220 (2005).

    Article  CAS  Google Scholar 

  30. Havens, C. G. & Walter, J. C. Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase. Gen. Dev. 25, 1568–1582 (2011).

    Article  CAS  Google Scholar 

  31. Havens, C. G. & Walter, J. C. Docking of a specialized PIP Box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Mol. Cell 35, 93–104 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ong, S. E., Foster, L. J. & Mann, M. Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–130 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. MacCoss, M. J., Wu, C. C., Matthews, D. E. & Yates, J. R. 3rd. Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides. Anal. Chem. 77, 7646–7653 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Nielsen, M. L. et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods 5, 459–460 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Wagner, S. A. et al. Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol. Cell Proteomicshttp://dx.doi.org/10.1074/mcp.M112.017905 (2012).

  36. Michalski, A. et al. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell Proteomics 10, M111 011015 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Olsen, J. V. et al. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cell Proteomics 8, 2759–2769 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kelstrup, C. D., Young, C., Lavallee, R., Nielsen, M. L. & Olsen, J. V. Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. J. Proteome Res. 11, 3487–3497 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Olsen, J. V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4, 709–712 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  PubMed  Google Scholar 

  42. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Craig, R. & Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Szklarczyk, D. et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 39, D561–D568 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P. L. & Ideker, T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27, 431–432 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell Proteomics 10, M110 003590 (2011).

    Article  PubMed  Google Scholar 

  49. 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 

  50. Mailand, N., Bekker-Jensen, S., Bartek, J. & Lukas, J. Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress. Mol. Cell 23, 307–318 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Maya-Mendoza, A., Petermann, E., Gillespie, D. A., Caldecott, K. W. & Jackson, D. A. Chk1 regulates the density of active replication origins during the vertebrate S phase. EMBO J. 26, 2719–2731 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Parris, C. N. & Seidman, M. M. A signature element distinguishessibling and independent mutations in a shuttle vector plasmid. Gene 117, 1–5 (1992).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to R. Schwab and W. Niedzwiedz for help with setting up the DNA fibre assay, and we thank A. Lehmann and M. Seidman for providing reagents. This work was supported by a generous donation from the Novo Nordisk Foundation, and grants from the Danish Medical Research Council (FSS10-081753 and FSS10-083519), the Danish Cancer Society, the Lundbeck Foundation and the European Commission seventh Framework Programme (PRIME-XS). P.B. and S.A.W. are supported by postdoctoral grants from the Danish Research Council (FSS12-12610, FSS10-085134). The Center for Protein Research is supported by a generous grant from the Novo Nordisk Foundation.

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L.K.P. performed most of the cell- and biochemistry-based experiments, supported by S.L.P. P.B. and S.A.W. carried out global, quantitative ubiquitylation analysis and analysed the data. K.B.S., J.W.P. and M.L.N. helped with the identification of PAF15 ubiquitylation sites and initial MS analyses. S.B-J., N.M. and C.C. supervised the project and analysed data. N.M. and C.C. wrote the paper.

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Correspondence to Niels Mailand or Chunaram Choudhary.

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Povlsen, L., Beli, P., Wagner, S. et al. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat Cell Biol 14, 1089–1098 (2012). https://doi.org/10.1038/ncb2579

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