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SUMOylation regulates Rad18-mediated template switch

Abstract

Replication by template switch is thought to mediate DNA damage-bypass and fillings of gaps. Gap-filling repair requires homologous recombination as well as Rad18- and Rad5-mediated proliferating cell nuclear antigen (PCNA) polyubiquitylation. However, it is unclear whether these processes are coordinated, and the physical evidence for Rad18–Rad5-dependent template switch at replication forks is still elusive. Here we show, using genetic and physical approaches, that in budding yeast (Saccharomyces cerevisiae) Rad18 is required for the formation of X-shaped sister chromatid junctions (SCJs) at damaged replication forks through a process involving PCNA polyubiquitylation and the ubiquitin-conjugating enzymes Mms2 and Ubc13. The Rad18–Mms2-mediated damage-bypass through SCJs requires the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 and SUMOylated PCNA, and is coordinated with Rad51-dependent recombination events. We propose that the Rad18–Rad5–Mms2-dependent SCJs represent template switch events. Altogether, our results unmask a role for PCNA ubiquitylation and SUMOylation pathways in promoting transient damage-induced replication-coupled recombination events involving sister chromatids at replication forks.

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Figure 1: Rad18 promotes SCJ formation during replication of damaged templates, but its functionality requires SUMOylation.
Figure 2: Sgs1 functions to resolve the damage-induced SCJs formed by Rad18-Mms2-PCNA polyubiquitylation.
Figure 3: The contribution of PCNA SUMOylation in the formation of SCJs accumulating in sgs1 during replication of damaged templates.
Figure 4: Two pathways contributing to SCJ formation.

References

  1. Prakash, L. Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol. Gen. Genet. 184, 471–478 (1981)

    Article  CAS  Google Scholar 

  2. Lopes, M., Foiani, M. & Sogo, J. M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27 (2006)

    Article  CAS  Google Scholar 

  3. Branzei, D. & Foiani, M. Interplay of replication checkpoints and repair proteins at stalled replication forks. DNA Repair (Amst.) 6, 994–1003 (2007)

    Article  CAS  Google Scholar 

  4. Lehmann, A. R. & Fuchs, R. P. Gaps and forks in DNA replication: Rediscovering old models. DNA Repair (Amst.) 5, 1495–1498 (2006)

    Article  CAS  Google Scholar 

  5. Fabre, F., Chan, A., Heyer, W. D. & Gangloff, S. Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl Acad. Sci. USA 99, 16887–16892 (2002)

    Article  ADS  CAS  Google Scholar 

  6. Symington, L. S. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66, 630–670 (2002)

    Article  CAS  Google Scholar 

  7. San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 (2008)

    Article  CAS  Google Scholar 

  8. Gangavarapu, V., Prakash, S. & Prakash, L. Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae . Mol. Cell. Biol. 27, 7758–7764 (2007)

    Article  CAS  Google Scholar 

  9. Zhang, H. & Lawrence, C. W. The error-free component of the RAD6/RAD18 DNA damage tolerance pathway of budding yeast employs sister-strand recombination. Proc. Natl Acad. Sci. USA 102, 15954–15959 (2005)

    Article  ADS  CAS  Google Scholar 

  10. Torres-Ramos, C. A., Prakash, S. & Prakash, L. Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae . Mol. Cell. Biol. 22, 2419–2426 (2002)

    Article  CAS  Google Scholar 

  11. Haracska, L. et al. Opposing effects of ubiquitin conjugation and SUMO modification of PCNA on replicational bypass of DNA lesions in Saccharomyces cerevisiae . Mol. Cell. Biol. 24, 4267–4274 (2004)

    Article  CAS  Google Scholar 

  12. Higgins, N. P., Kato, K. & Strauss, B. A model for replication repair in mammalian cells. J. Mol. Biol. 101, 417–425 (1976)

    Article  CAS  Google Scholar 

  13. Goldfless, S. J. et al. DNA repeat rearrangements mediated by DnaK-dependent replication fork repair. Mol. Cell 21, 595–604 (2006)

    Article  CAS  Google Scholar 

  14. Branzei, D. & Foiani, M. Template switching: from replication fork repair to genome rearrangements. Cell 131, 1228–1230 (2007)

    Article  CAS  Google Scholar 

  15. Jentsch, S., McGrath, J. P. & Varshavsky, A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329, 131–134 (1987)

    Article  ADS  CAS  Google Scholar 

  16. 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  Google Scholar 

  17. Ulrich, H. D. & Jentsch, S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 19, 3388–3397 (2000)

    Article  CAS  Google Scholar 

  18. Bailly, V. et al. Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev. 8, 811–820 (1994)

    Article  CAS  Google Scholar 

  19. Branzei, D. & Foiani, M. Regulation of DNA repair throughout the cell cycle. Nature Rev. Mol. Cell Biol. 4, 297–308 (2008)

    Article  Google Scholar 

  20. Hoege, C. et al. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002)

    Article  ADS  CAS  Google Scholar 

  21. Stelter, P. & Ulrich, H. D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191 (2003)

    Article  ADS  CAS  Google Scholar 

  22. Kannouche, P. L., Wing, J. & Lehmann, A. R. Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol. Cell 14, 491–500 (2004)

    Article  CAS  Google Scholar 

  23. Geiss-Friedlander, R. & Melchior, F. Concepts in sumoylation: a decade on. Nature Rev. Mol. Cell Biol. 8, 947–956 (2007)

    Article  CAS  Google Scholar 

  24. Pfander, B. et al. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436, 428–433 (2005)

    Article  ADS  CAS  Google Scholar 

  25. Papouli, E. et al. Crosstalk between SUMO and Ubiquitin on PCNA Is Mediated by Recruitment of the Helicase Srs2p. Mol. Cell 19, 123–133 (2005)

    Article  CAS  Google Scholar 

  26. Lawrence, C. W. & Christensen, R. B. Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. J. Bacteriol. 139, 866–876 (1979)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Aboussekhra, A. et al. RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res. 17, 7211–7219 (1989)

    Article  CAS  Google Scholar 

  28. Schiestl, R. H., Prakash, S. & Prakash, L. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics 124, 817–831 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Palladino, F. & Klein, H. L. Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics 132, 23–37 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Krejci, L. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305–309 (2003)

    Article  ADS  CAS  Google Scholar 

  31. Veaute, X. et al. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423, 309–312 (2003)

    Article  ADS  CAS  Google Scholar 

  32. Branzei, D. et al. Ubc9- and Mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127, 509–522 (2006)

    Article  CAS  Google Scholar 

  33. Liberi, G. et al. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev. 19, 339–350 (2005)

    Article  CAS  Google Scholar 

  34. Mankouri, H. W., Ngo, H. P. & Hickson, I. D. Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3. Mol. Biol. Cell 18, 4062–4073 (2007)

    Article  CAS  Google Scholar 

  35. Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003)

    Article  ADS  CAS  Google Scholar 

  36. Johnson, E. S. Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382 (2004)

    Article  CAS  Google Scholar 

  37. Onoda, F., Seki, M., Miyajima, A. & Enomoto, T. Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae . Mol. Gen. Genet. 264, 702–708 (2001)

    Article  CAS  Google Scholar 

  38. Branzei, D., Seki, M. & Enomoto, T. Rad18/Rad5/Mms2-mediated polyubiquitination of PCNA is implicated in replication completion during replication stress. Genes Cells 9, 1031–1042 (2004)

    Article  CAS  Google Scholar 

  39. Tateishi, S. et al. Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc. Natl Acad. Sci. USA 97, 7927–7932 (2000)

    Article  ADS  CAS  Google Scholar 

  40. 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  Google Scholar 

  41. Motegi, A. et al. Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination. J. Cell Biol. 175, 703–708 (2006)

    Article  CAS  Google Scholar 

  42. Motegi, A. et al. Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae . Mol. Cell. Biol. 26, 1424–1433 (2006)

    Article  CAS  Google Scholar 

  43. Daee, D. L., Mertz, T. & Lahue, R. S. Postreplication repair inhibits CAG·CTG repeat expansions in Saccharomyces cerevisiae . Mol. Cell. Biol. 27, 102–110 (2007)

    Article  CAS  Google Scholar 

  44. Johnson, R. E. et al. Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. Mol. Cell. Biol. 12, 3807–3818 (1992)

    Article  CAS  Google Scholar 

  45. Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nature Rev. Mol. Cell Biol. 7, 739–750 (2006)

    Article  CAS  Google Scholar 

  46. Otsuki, M. et al. Functional interactions between BLM and XRCC3 in the cell. J. Cell Biol. 179, 53–63 (2007)

    Article  CAS  Google Scholar 

  47. Szuts, D. et al. Role for RAD18 in homologous recombination in DT40 cells. Mol. Cell. Biol. 26, 8032–8041 (2006)

    Article  CAS  Google Scholar 

  48. Tateishi, S. et al. Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells. Mol. Cell. Biol. 23, 474–481 (2003)

    Article  CAS  Google Scholar 

  49. Yamashita, Y. M. et al. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J. 21, 5558–5566 (2002)

    Article  CAS  Google Scholar 

  50. Shekhar, M. P. et al. Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation. Cancer Res. 62, 2115–2124 (2002)

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S. Jentsch and H. Ulrich for yeast strains, T. Enomoto for critical reading, and all members of our laboratories for helpful discussions. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro to D.B., Association for International Cancer Research and European Community GENICA grant to M.F. and D.B., and partly by European Community DNA Repair grant, Telethon, MIUR, and Ministry of Health to M.F. D.B. was partly supported by the Buzzati-Traverso foundation.

Author Contributions D.B. conceived the project, designed and performed the experiments, and wrote the paper. F.V. quantified the 2D gels, performed data analysis and provided technical help. M.F. discussed the results, analysed the data, edited the manuscript and provided scientific advice and financial support.

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Correspondence to Dana Branzei.

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Branzei, D., Vanoli, F. & Foiani, M. SUMOylation regulates Rad18-mediated template switch. Nature 456, 915–920 (2008). https://doi.org/10.1038/nature07587

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