Ribonucleotide incorporation by yeast DNA polymerase ζ

DNA Repair (Amst). 2014 Jun;18:63-7. doi: 10.1016/j.dnarep.2014.02.017. Epub 2014 Mar 24.

Abstract

During replication in yeast, the three B family DNA replicases frequently incorporate ribonucleotides (rNMPs) into DNA, and their presence in the nuclear genome can affect genome stability. This prompted us to examine ribonucleotide incorporation by the fourth B family member, Pol ζ, the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol ζ inserts rNMPs into DNA and can extend primer termini containing 3'-ribonucleotides. We then measure rNMP incorporation by Pol ζ in the presence of its cofactors, RPA, RFC and PCNA and at normal cellular dNTP and rNTP concentrations that exist under unstressed conditions. Under these conditions, Pol ζ stably incorporates one rNMP for every 200-300 dNMPs incorporated, a frequency that is slightly higher than for the high fidelity replicative DNA polymerases. Under damage-induced conditions wherein cellular dNTP concentrations are elevated 5-fold, Pol ζ only incorporates one rNMP per 1300 dNMPs. Functional interaction of Pol ζ with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol ζ-mediated mutagenesis in vivo may be rare.

Keywords: DNA polymerase; Mutagenesis; Ribonucleotides; Translesion synthesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • DNA Damage
  • DNA Replication
  • DNA, Fungal / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyribonucleotides / metabolism
  • Mutation
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism
  • Replication Protein A / metabolism
  • Ribonucleotides / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • DNA, Fungal
  • Deoxyribonucleotides
  • REV7 protein, S cerevisiae
  • RFA1 protein, S cerevisiae
  • Replication Protein A
  • Ribonucleotides
  • Saccharomyces cerevisiae Proteins
  • DNA polymerase zeta
  • Nucleotidyltransferases
  • REV1 protein, S cerevisiae
  • DNA-Directed DNA Polymerase
  • REV3 protein, S cerevisiae