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.
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