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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References
Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006).
Deshaies, R. J. & Joazeiro, C. A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).
Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).
Ciccia, A. & Elledge, S. J. The DNA damage response: Making it safe to play with knives. Mol. Cell 40, 179–204 (2010).
Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).
Al-Hakim, A. et al. The ubiquitous role of ubiquitin in the DNA damage response. DNA Repair (Amst) 9, 1229–1240 (2010).
Ulrich, H. D. & Walden, H. Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489 (2010).
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).
Bergink, S. & Jentsch, S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 458, 461–467 (2009).
Lehmann, A. R. et al. Translesion synthesis: Y-family polymerases and the polymerase switch. DNA Repair (Amst) 6, 891–899 (2007).
Moldovan, G. L., Pfander, B. & Jentsch, S. PCNA, the maestro of the replication fork. Cell 129, 665–679 (2007).
Friedberg, E. C. Suffering in silence: the tolerance of DNA damage. Nat. Rev. Mol. Cell Biol. 6, 943–953 (2005).
Bienko, M. et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, 1821–1824 (2005).
Huang, T. T. et al. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat. Cell Biol. 8, 339–347 (2006).
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).
Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).
Emanuele, M. J. et al. Global identification of modular cullin-RING ligase substrates. Cell 147, 459–474 (2011).
Xu, G., Paige, J. S. & Jaffrey, S. R. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat. Biotechnol. 28, 868–873 (2010).
Gillet, L. C. & Scharer, O. D. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem. Rev. 106, 253–276 (2006).
Sugasawa, K. et al. UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex. Cell 121, 387–400 (2005).
Xiao, A. et al. WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity. Nature 457, 57–62 (2009).
Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).
Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401–1412 (2011).
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).
Williamson, A. et al. Regulation of ubiquitin chain initiation to control the timing of substrate degradation. Mol. Cell 42, 744–757 (2011).
Abbas, T. & Dutta, A. CRL4Cdt2: master coordinator of cell cycle progression and genome stability. Cell Cycle 10, 241–249 (2011).
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).
Niimi, A. et al. Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells. Proc. Natl Acad. Sci. USA 105, 16125–16130 (2008).
Choi, J. H. & Pfeifer, G. P. The role of DNA polymerase eta in UV mutational spectra. DNA Repair (Amst) 4, 211–220 (2005).
Havens, C. G. & Walter, J. C. Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase. Gen. Dev. 25, 1568–1582 (2011).
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).
Ong, S. E., Foster, L. J. & Mann, M. Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–130 (2003).
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).
Nielsen, M. L. et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods 5, 459–460 (2008).
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).
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).
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).
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).
Olsen, J. V. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4, 709–712 (2007).
Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
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).
Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).
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).
Craig, R. & Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467 (2004).
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).
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).
Danielsen, J. M. et al. Mass spectrometric analysis of lysine ubiquitylation reveals promiscuity at site level. Mol. Cell Proteomics 10, M110 003590 (2011).
Mailand, N. et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 131, 887–900 (2007).
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).
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).
Parris, C. N. & Seidman, M. M. A signature element distinguishessibling and independent mutations in a shuttle vector plasmid. Gene 117, 1–5 (1992).
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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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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DOI: https://doi.org/10.1038/ncb2579
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