Ribonucleotide incorporation by yeast DNA polymerase ζ

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 zeta, the enzyme responsible for the majority of damage-induced mutagenesis in eukaryotes. We first show that Pol inserts rNMPs into DNA and can extend primer termini containing 3'-ribonucleotides. We then measure rNMP incorporation by Pol 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 zeta 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 zeta only incorporates one rNMP per 1300 dNMPs. Functional interaction of Pol zeta with the mutasome assembly factor Rev1 gives comparable rNMP incorporation frequencies. These results suggest that ribonucleotide incorporation into DNA during Pol zeta-mediated mutagenesis in vivo may be rare. (C) 2014 Elsevier B.V. All rights reserved.