Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis

Lajos Haracska, Ildiko Unk, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticlepeer-review

106 Scopus citations


The Rad6-Rad18 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) η- and ζ-dependent translesion DNA synthesis (TLS) and a Rad5-Mms2-Ubc13-dependent pathway. In DNA-damaged yeast cells proliferating cell nuclear antigen (PCNA) becomes monoubiquitylated at the K164 residue, and genetic studies in yeast have indicated a requirement for this modification in TLS mediated by Polη and Polζ. To be able to decipher the role of PCNA monoubiquitylation in the TLS process, we have reconstituted this PCNA modification in vitro from purified yeast proteins. We show that, in addition to the requirement for Rad6-Rad18, the reaction depends on the loading of the PCNA homotrimeric ring onto the DNA by replication factor C and that all three PCNA monomers become efficiently ubiquitylated. The availability of PCNA monoubiquitylated on all of its three monomers has enabled us to examine the effects of this PCNA modification on DNA synthesis by Pols δ, η, ζ, and Rev1. Contrary to the prevailing ideas that presume a role for PCNA ubiquitylation in the disruption of Polo's binding to PCNA or in the enhancement of the binding affinity of the TLS Pols for PCNA, we find that PCNA ubiquitylation does not affect any of these processes. These observations lead us to suggest a role for PCNA monoubiquitylation in disrupting the PCNA binding of a protein(s) that otherwise is inhibitory to the binding of PCNA by TLS Pols.

Original languageEnglish (US)
Pages (from-to)6477-6482
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number17
StatePublished - Apr 25 2006


  • DNA polymerase ζ
  • DNA polymerase η
  • Proliferating cell nuclear antigen ubiquitylation
  • Rad6-Rad18 ubiquitin-conjugating enzyme
  • Rev1

ASJC Scopus subject areas

  • General


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