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

Ribonucleotide incorporation by yeast DNA polymerase ζ.

Author information

1
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
2
Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
3
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: burgers@biochem.wustl.edu.

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

PMID:
24674899
PMCID:
PMC4402711
DOI:
10.1016/j.dnarep.2014.02.017
[Indexed for MEDLINE]
Free PMC Article

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