Detection of PCNA Modifications in Saccharomyces cerevisiae

  • Adelina A. Davies
  • Helle D. UlrichEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 920)


PCNA modifications by members of the ubiquitin family are associated with a range of different transactions during replication of damaged and undamaged DNA. This chapter describes detailed protocols for the detection and isolation of ubiquitin and SUMO conjugates of PCNA from total budding yeast cell lysates, using Ni-NTA affinity chromatography under denaturing conditions. We describe approaches based on the purification of PCNA itself and on the isolation of total ubiquitin or SUMO conjugates. The chapter covers the construction of the appropriate strains, methods for the detection of modified PCNA, and the use of various DNA-damaging agents as well as mutants of PCNA and relevant conjugation enzymes to examine the cellular response to replication stress.

Key words

PCNA Ubiquitin SUMO DNA damage bypass Postreplication repair Ni-NTA affinity chromatography Budding yeast 



The authors would like to thank H. Windecker for contributing images of PCNA conjugates and P. Burgers for providing plasmid pBL243. Work in this lab is supported by Cancer Research UK.


  1. 1.
    Hoeijmakers JH (2009) DNA damage, aging, and cancer. N Engl J Med 361:1475–1485PubMedCrossRefGoogle Scholar
  2. 2.
    Lawrence C (1994) The RAD6 DNA repair pathway in Saccharomyces cerevisiae: what does it do, and how does it do it? Bioessays 16:253–258PubMedCrossRefGoogle Scholar
  3. 3.
    Friedberg EC (2005) Suffering in silence: the tolerance of DNA damage. Nat Rev Mol Cell Biol 6:943–953PubMedCrossRefGoogle Scholar
  4. 4.
    Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11:208–219PubMedCrossRefGoogle Scholar
  5. 5.
    Lehmann AR, Niimi A, Ogi T, Brown S, Sabbioneda S, Wing JF, Kannouche PL, Green CM (2007) Translesion synthesis: Y-family polymerases and the polymerase switch. DNA Repair 6:891–899PubMedCrossRefGoogle Scholar
  6. 6.
    Waters LS, Minesinger BK, Wiltrout ME, D’Souza S, Woodruff RV, Walker GC (2009) Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance. Microbiol Mol Biol Rev 73:134–154PubMedCrossRefGoogle Scholar
  7. 7.
    Ulrich HD (2011) Timing and spacing of ubiquitin-dependent DNA damage bypass. FEBS Lett 585(18):2861–2867PubMedCrossRefGoogle Scholar
  8. 8.
    Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S (2002) RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419:135–141PubMedCrossRefGoogle Scholar
  9. 9.
    Stelter P, Ulrich HD (2003) Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425:188–191PubMedCrossRefGoogle Scholar
  10. 10.
    Watanabe K, Tateishi S, Kawasuji M, Tsurimoto T, Inoue H, Yamaizumi M (2004) Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J 23:3886–3896PubMedCrossRefGoogle Scholar
  11. 11.
    Bienko M, Green CM, Crosetto N, Rudolf F, Zapart G, Coull B, Kannouche P, Wider G, Peter M, Lehmann AR, Hofmann K, Dikic I (2005) Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310:1821–1824PubMedCrossRefGoogle Scholar
  12. 12.
    Guo C, Tang TS, Bienko M, Parker JL, Bielen AB, Sonoda E, Takeda S, Ulrich HD, Dikic I, Friedberg EC (2006) Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage. Mol Cell Biol 26:8892–8900PubMedCrossRefGoogle Scholar
  13. 13.
    Plosky BS, Vidal AE, de Henestrosa AR, McLenigan MP, McDonald JP, Mead S, Woodgate R (2006) Controlling the subcellular localization of DNA polymerases ι and η via interactions with ubiquitin. EMBO J 25:2847–2855PubMedCrossRefGoogle Scholar
  14. 14.
    Parker JL, Bielen AB, Dikic I, Ulrich HD (2007) Contributions of ubiquitin- and PCNA-binding domains to the activity of polymerase η in Saccharomyces cerevisiae. Nucleic Acids Res 35:881–889PubMedCrossRefGoogle Scholar
  15. 15.
    Parker JL, Ulrich HD (2009) Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5. EMBO J 28:3657–3666PubMedCrossRefGoogle Scholar
  16. 16.
    Davies AA, Huttner D, Daigaku Y, Chen S, Ulrich HD (2008) Activation of ubiquitin-dependent DNA damage bypass is mediated by Replication Protein A. Mol Cell 29:625–636PubMedCrossRefGoogle Scholar
  17. 17.
    Niimi A, Brown S, Sabbioneda S, Kannouche PL, Scott A, Yasui A, Green CM, Lehmann AR (2008) Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells. Proc Natl Acad Sci U S A 105:16125–16130PubMedCrossRefGoogle Scholar
  18. 18.
    Ulrich HD (2009) Regulating post-translational modifications of the eukaryotic replication clamp PCNA. DNA Repair 8:461–469PubMedCrossRefGoogle Scholar
  19. 19.
    Parker JL, Bucceri A, Davies AA, Heidrich K, Windecker H, Ulrich HD (2008) SUMO modification of PCNA is controlled by DNA. EMBO J 27:2422–2431PubMedCrossRefGoogle Scholar
  20. 20.
    Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P, Ulrich HD (2005) Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol Cell 19:123–133PubMedCrossRefGoogle Scholar
  21. 21.
    Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S (2005) SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436:428–433PubMedGoogle Scholar
  22. 22.
    Moldovan GL, Pfander B, Jentsch S (2006) PCNA controls establishment of sister chromatid cohesion during S phase. Mol Cell 23:723–732PubMedCrossRefGoogle Scholar
  23. 23.
    Parnas O, Zipin-Roitman A, Pfander B, Liefshitz B, Mazor Y, Ben-Aroya S, Jentsch S, Kupiec M (2010) Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA. EMBO J 29:2611–2622PubMedCrossRefGoogle Scholar
  24. 24.
    Panse VG, Hardeland U, Werner T, Kuster B, Hurt E (2004) A proteome-wide approach identifies sumoylated substrate proteins in yeast. J Biol Chem 279:41346–41351PubMedCrossRefGoogle Scholar
  25. 25.
    Vertegaal AC, Ogg SC, Jaffray E, Rodriguez MS, Hay RT, Andersen JS, Mann M, Lamond AI (2004) A proteomic study of SUMO-2 target proteins. J Biol Chem 279:33791–33798PubMedCrossRefGoogle Scholar
  26. 26.
    Wohlschlegel JA, Johnson ES, Reed SI, Yates JR 3rd (2004) Global analysis of protein sumoylation in Saccharomyces cerevisiae. J Biol Chem 279:45662–45668PubMedCrossRefGoogle Scholar
  27. 27.
    Zhao Y, Kwon SW, Anselmo A, Kaur K, White MA (2004) Broad spectrum identification of cellular small ubiquitin-related modifier (SUMO) substrate proteins. J Biol Chem 279:20999–21002PubMedCrossRefGoogle Scholar
  28. 28.
    Zhou W, Ryan JJ, Zhou H (2004) Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J Biol Chem 279:32262–32268PubMedCrossRefGoogle Scholar
  29. 29.
    Denison C, Rudner AD, Gerber SA, Bakalarski CE, Moazed D, Gygi SP (2005) A proteomic strategy for gaining insights into protein sumoylation in yeast. Mol Cell Proteomics 4:246–254PubMedCrossRefGoogle Scholar
  30. 30.
    Hannich JT, Lewis A, Kroetz MB, Li SJ, Heide H, Emili A, Hochstrasser M (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J Biol Chem 280:4102–4110PubMedCrossRefGoogle Scholar
  31. 31.
    Kirkpatrick DS, Weldon SF, Tsaprailis G, Liebler DC, Gandolfi AJ (2005) Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. Proteomics 5:2104–2111PubMedCrossRefGoogle Scholar
  32. 32.
    Peng J, Cheng D (2005) Proteomic analysis of ubiquitin conjugates in yeast. Methods Enzymol 399:367–381PubMedCrossRefGoogle Scholar
  33. 33.
    Rosas-Acosta G, Russell WK, Deyrieux A, Russell DH, Wilson VG (2005) A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers. Mol Cell Proteomics 4:56–72PubMedGoogle Scholar
  34. 34.
    Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular biology, vol 194. Academic, San DiegoCrossRefGoogle Scholar
  35. 35.
    Ayyagari R, Impellizzeri KJ, Yoder BL, Gary SL, Burgers PM (1995) A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol Cell Biol 15:4420–4429PubMedGoogle Scholar
  36. 36.
    Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534PubMedCrossRefGoogle Scholar
  37. 37.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  38. 38.
    Windecker H, Ulrich HD (2007) Architecture and assembly of poly-SUMO chains on PCNA in Saccharomyces cerevisiae. J Mol Biol 376:221–231PubMedCrossRefGoogle Scholar
  39. 39.
    Ulrich HD, Davies AA (2009) In vivo detection and characterization of sumoylation targets in Saccharomyces cerevisiae. Methods Mol Biol 497:81–103PubMedCrossRefGoogle Scholar
  40. 40.
    Finley D, Ozkaynak E, Varshavsky A (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48:1035–1046PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Clare Hall LaboratoriesCancer Research UK London Research InstituteSouth MimmsUK

Personalised recommendations