Saccharomyces cerevisiae DNA polymerase delta : High fidelity for base substitutions but lower fidelity for single-and multi-base deletions

doi:10.1074/jbc.M505236200

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Saccharomyces cerevisiae DNA polymerase $delta$ : High fidelity for base substitutions but lower fidelity for single-and multi-base deletions

John M. Fortune, Youri I. Pavlov, Carrie M. Welch, Erik Johansson, Peter M. J. Burgers, and Thomas A. Kunkel

National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

Corresponding Author: kunkel{at}niehs.nih.gov

Eukaryotic DNA polymerase $delta$ (Pol $delta$ ) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion of individual dNTPs opposite a template guanine, Pol $delta$ is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3´-exonuclease activity, implies that Pol $delta$ should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol $delta$ does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations per 100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol $delta$ confirm the high nucleotide selectivity of the polymerase and further indicate that proofreading enhances the base substitution fidelity of the wild type enzyme by at least 60-fold. However, Pol $delta$ inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions per 100,000 nucleotides polymerized. Moreover, wild type Pol $delta$ frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol $delta$ and Pol $epsilon$ both have high base substitution fidelity, Pol $delta$ is much less accurate than Pol $epsilon$ for deletions involving repetitive sequences. Thus, strand slippage during replication by wild-type Pol $delta$ may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.

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Saccharomyces cerevisiae DNA polymerase : High fidelity for base substitutions but lower fidelity for single-and multi-base deletions

Saccharomyces cerevisiae DNA polymerase $delta$ : High fidelity for base substitutions but lower fidelity for single-and multi-base deletions