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J. Biol. Chem., Vol. 280, Issue 33, 29980-29987, August 19, 2005

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Saccharomyces cerevisiae DNA Polymerase

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**

From the Laboratory of Molecular Genetics and Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the^¶Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, Missouri 63110, and the ^||Department of Medical Biochemistry and Biophysics, Umeä University, Umeä SE-901 87, Sweden

Eukaryotic DNA polymerase (Pol ) 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 is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3'-exonuclease activity, implies that Pol should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations/100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol 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 inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions/100,000 nucleotides polymerized. Moreover, wild type Pol frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol and Pol both have high base substitution fidelity, Pol is much less accurate than Pol for deletions involving repetitive sequences. Thus, strand slippage during replication by wild type Pol may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.

Received for publication, May 12, 2005 , and in revised form, June 13, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198.

^** To whom correspondence should be addressed: NIEHS, National Institutes of Health, P. O. Box 12233, MD E3-01, Research Triangle Park, NC 27709. Tel.: 919-541-2644; Fax: 919-541-7593; E-mail: kunkel{at}niehs.nih.gov.

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