The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ

PLoS Genet. 2017 Dec 27;13(12):e1007119. doi: 10.1371/journal.pgen.1007119. eCollection 2017 Dec.

Abstract

Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • DNA / genetics
  • DNA / metabolism
  • DNA Damage
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Repair
  • DNA Replication
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Models, Genetic
  • Mutation
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Binding
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Ubiquitination
  • Ultraviolet Rays

Substances

  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • DNA
  • DNA polymerase zeta
  • Nucleotidyltransferases
  • REV1 protein, S cerevisiae
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase

Grant support

The research was supported by the Agence Nationale de la Recherche (ANR-09-PIRI-0015-03 and ANR-10-INBS-05), by the Centre national de la recherche scientifique (recurrent funding) and the Commissariat aux Energies Atomiques et Alternatives (recurrent funding). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.