PCNA monoubiquitylation and DNA polymerase eta ubiquitin-binding domain are required to prevent 8-oxoguanine-induced mutagenesis in Saccharomyces cerevisiae

Nucleic Acids Res. 2009 May;37(8):2549-59. doi: 10.1093/nar/gkp105. Epub 2009 Mar 5.

Abstract

7,8-Dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic DNA lesion. In Saccharomyces cerevisiae, the 8-oxoG DNA N-glycosylase (Ogg1) acts as the primary defense against 8-oxoG. Here, we present evidence for cooperation between Rad18-Rad6-dependent monoubiquitylation of PCNA at K164, the damage-tolerant DNA polymerase eta and the mismatch repair system (MMR) to prevent 8-oxoG-induced mutagenesis. Preventing PCNA modification at lysine 164 (pol30-K164R) results in a dramatic increase in GC to TA mutations due to endogenous 8-oxoG in Ogg1-deficient cells. In contrast, deletion of RAD5 or SIZ1 has little effect implying that the modification of PCNA relevant for preventing 8-oxoG-induced mutagenesis is monoubiquitin as opposed to polyubiquitin or SUMO. We also report that the ubiquitin-binding domain (UBZ) of Pol eta is essential to prevent 8-oxoG-induced mutagenesis but only in conjunction with a functional PCNA-binding domain (PIP). We propose that PCNA is ubiquitylated during the repair synthesis reaction after the MMR-dependent excision of adenine incorporated opposite to 8-oxoG. Monoubiquitylation of PCNA would favor the recruitment of Pol eta thereby allowing error-free incorporation of dCMP opposite to 8-oxoG. This study suggests that Pol eta and the post-replication repair (PRR) machinery can also prevent mutagenesis at DNA lesions that do not stall replication forks.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Canavanine / pharmacology
  • DNA Glycosylases / genetics
  • DNA Glycosylases / metabolism
  • DNA Repair
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Gene Deletion
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Mutagenesis*
  • Mutation
  • Proliferating Cell Nuclear Antigen / chemistry
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Ubiquitin / metabolism
  • Ubiquitination*

Substances

  • Pol32 protein, S cerevisiae
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Canavanine
  • 8-hydroxyguanine
  • Guanine
  • DNA-Directed DNA Polymerase
  • Rad30 protein
  • DNA Glycosylases
  • Ogg1 protein, S cerevisiae
  • mutY adenine glycosylase