Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107

Nature Cell Biology volume 12, pages 74–79 (2010)Cite this article

Abstract

In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalysed by DNA ligase I (ref. 1). An individual with a DNA ligase I deficiency exhibits growth retardation, sunlight sensitivity and severe immunosuppression2, probably due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. As DNA replication and DDR pathways are highly conserved in eukaryotes, we used Saccharomyces cerevisiae as a model system to address this issue. We uncovered a new pathway, which facilitates ubiquitylation at Lys 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitylation at Lys 164 of PCNA in response to UV irradiation, which triggers translesion synthesis3, modification of Lys 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad6 (ref. 4) nor the ubiquitin ligase (E3) Rad18 (ref. 5), but requires the E2 variant Mms2 (ref. 6) in conjunction with Ubc4 (ref. 7) and the E3 Rad5 (Refs 8, 9). Surprisingly, DNA ligase I-deficient S. cerevisiae cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitylation of PCNA in response to DNA ligase I deficiency is conserved in humans, yet the lysine residue that is modified remains to be determined. We propose that PCNA ubiquitylation provides a 'DNA damage code' that allows cells to categorize different types of defects that arise during DNA replication.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: DNA ligase I is required for S phase progression.
Figure 2: S. cerevisiae PCNA is mono-ubiquitylated in cdc9 mutants.
Figure 3: PCNA mono-ubiquitylation in cdc9 mutants is mediated by Mms2, Rad5 and Ubc4 but not Ubc13.
Figure 4: Mms2 but not Ubc13 is required for S phase checkpoint activation in cdc9 mutants.
Figure 5: PCNA mono-ubiquitylation occurs at Lys 107 in DNA ligase I mutants and is required for Rad53 activation.

Similar content being viewed by others

References

  1. Ellenberger, T. & Tomkinson, A. E. Eukaryotic DNA ligases: structural and functional insights. Annu. Rev. Biochem 77, 313–338 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Webster, A. D., Barnes, D. E., Arlett, C. F., Lehmann, A. R. & Lindahl, T. Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene. Lancet 339, 1508–1509 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Bailly, V., Lauder, S., Prakash, S. & Prakash, L. Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities. J. Biol. Chem. 272, 23360–23365 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Broomfield, S., Chow, B. L. & Xiao, W. MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc. Natl Acad. Sci. USA 95, 5678–5683 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Seufert, W. & Jentsch, S. Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 9, 543–550 (1990).

    Article  CAS  PubMed  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. Schiestl, R. H., Reynolds, P., Prakash, S. & Prakash, L. Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage. Mol. Cell. Biol. 9, 1882–1896 (1989).

    Article  CAS  PubMed  Google Scholar 

  11. Weinert, T. A. & Hartwell, L. H. Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. Genetics 134, 63–80 (1993).

    CAS  PubMed Central  PubMed  Google Scholar 

  12. Johnston, L. H. & Nasmyth, K. A. Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature 274, 891–893 (1978).

    Article  CAS  PubMed  Google Scholar 

  13. Dohmen, R. J., Wu, P. & Varshavsky, A. Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science 263, 1273–1276 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Bielinsky, A. K. & Gerbi, S. A. Chromosomal ARS1 has a single leading strand start site. Mol. Cell 3, 477–486 (1999).

    Article  CAS  PubMed  Google Scholar 

  15. Ireland, M. J., Reinke, S. S. & Livingston, D. M. The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast. Genetics 155, 1657–1665 (2000).

    CAS  PubMed Central  PubMed  Google Scholar 

  16. Osborn, A. J. & Elledge, S. J. Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev. 17, 1755–1767 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Sanchez, Y. et al. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271, 357–360 (1996).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  20. Haracska, L., Torres-Ramos, C. A., Johnson, R. E., Prakash, S. & Prakash, L. 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  PubMed  Google Scholar 

  21. 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 

  22. Zhang, Z., Shibahara, K. & Stillman, B. PCNA connects DNA replication to epigenetic inheritance in yeast. Nature 408, 221–225 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Ellison, M. J. & Hochstrasser, M. Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. J. Biol. Chem. 266, 21150–21157 (1991).

    CAS  PubMed  Google Scholar 

  24. Das-Bradoo, S., Ricke, R. M. & Bielinsky, A. K. Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast. Mol. Cell. Biol. 26, 4806–4817 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. van der Kemp, P. A., Padula, M. D., Burguiere-Slezak, G., Ulrich, H. D. & Boiteux, S. PCNA monoubiquitylation and DNA polymerase-eta ubiquitin-binding domain are required to prevent 8-oxoguanine-induced mutagenesis in Saccharomyces cerevisiae. Nucleic Acids Res. 37, 2549–2559 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, M. & Pickart, C. M. Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis. EMBO J. 24, 4324–4333 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Frampton, J. et al. Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol. Biol. Cell 17, 2976–2985 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. 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.

  30. Kao, H. I., Veeraraghavan, J., Polaczek, P., Campbell, J. L. & Bambara, R. A. On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing. J. Biol. Chem. 279, 15014–15024 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Labib, K., Tercero, J. A. & Diffley, J. F. Uninterrupted MCM2–7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Lorenz, M. C. et al. Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158, 113–117 (1995).

    Article  CAS  PubMed  Google Scholar 

  33. Wen, W., Meinkoth, J. L., Tsien, R. Y. & Taylor, S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Ricke, R. M. & Bielinsky, A. K. Mcm10 regulates the stability and chromatin association of DNA polymerase-α. Mol. Cell 16, 173–185 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Ricke, R. M. & Bielinsky, A. K. A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-α in budding yeast. J. Biol. Chem. 281, 18414–18425 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Diffley, J. F. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2–7 during G1 phase. Nature Cell Biol. 4, 198–207 (2002).

    Article  PubMed  Google Scholar 

  37. Silva, J. M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nature Genetics 37, 1281–1288 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Hannon, G. J. & Conklin, D. S. RNA interference by short hairpin RNAs expressed in vertebrate cells. Methods Mol. Biol. 257, 255–266 (2004).

    CAS  PubMed  Google Scholar 

  39. Motegi, A. et al. Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication fork. Proc. Natl Acad. Sci. USA 26, 12411–12416 (2008).

    Article  Google Scholar 

Download references

Acknowledgements

We thank D. J. Clarke, J. F. X. Diffley, D. Durocher, S. J. Elledge, M. Hochstrasser, S. Jentsch, D. Koepp and D. M. Livingston for strains and plasmids and J. F. X. Diffley, A. E. Tomkinson and Z. Zhang for antibodies against Rad53, Cdc9 and PCNA. We acknowledge the assistance of the Flow Cytometry Core Facility at the University of Minnesota. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). J.C.H. was supported by NIH training grant CA009138. This work was supported by a Grant-in-Aid from the University of Minnesota and NIH grant (GM074917) to A.K.B. who is a Scholar of the Leukemia and Lymphoma Society.

Author information

Author notes

  1. Sapna Das-Bradoo, Hai Dang Nguyen and Jamie L. Wood: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Minnesota, Molecular Biology and Biophysics, Minneapolis, 55455, MN, USA

    Sapna Das-Bradoo, Hai Dang Nguyen, Jamie L. Wood, Justin C. Haworth & Anja-Katrin Bielinsky

  2. Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, 55905, MN, USA

    Robin M. Ricke

Authors
  1. Sapna Das-Bradoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hai Dang Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jamie L. Wood
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robin M. Ricke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Justin C. Haworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anja-Katrin Bielinsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.D.-B. and H.D.N. conducted all PCNA ubiquitylation experiments in yeast, helped with data analysis and helped write the manuscript. J.L.W. conducted all experiments with human cells, helped with data analysis and helped write the manuscript. R.M.R conducted cell-cycle arrest and RIP mapping experiments, and helped with data analysis. J.C.H. helped with the construction of yeast mutants. A.-K.B. planned and supervised the project, and wrote the manuscript.

Corresponding author

Correspondence to Anja-Katrin Bielinsky.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1867 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Das-Bradoo, S., Nguyen, H., Wood, J. et al. Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107. Nat Cell Biol 12, 74–79 (2010). https://doi.org/10.1038/ncb2007

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2007

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing