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Originally published In Press as doi:10.1074/jbc.M204476200 on July 17, 2002

J. Biol. Chem., Vol. 277, Issue 40, 37422-37429, October 4, 2002
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Fidelity of DNA Polymerase epsilon  Holoenzyme from Budding Yeast Saccharomyces cerevisiae*

Kikuo ShimizuDagger §, Keiji HashimotoDagger , Jake M. Kirchner||, Wataru NakaiDagger , Hiroko NishikawaDagger , Michael A. Resnick, and Akio SuginoDagger **

From the Dagger  The Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan and the  Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

DNA polymerases delta  and epsilon  (pol delta  and epsilon ) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon , the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon  was 0.47 × 10-5 for G:G mismatches, 0.15 × 10-5 for T:G mismatches, and less than 0.01 × 10-5 for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon ; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon  mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon  is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.

* This work was supported in part by a grant-in-aid for scientific research on priority area (A), a grant-in-aid for scientific research (A) from the Ministry of Education, Science, Sports, and Culture of Japan, and Human Frontier Science Program Research Grant RG039/2000-M (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: The Radioisotope Research Center, Osaka University, 2-4 Yamada-oka, Suita, Osaka 565-0871, Japan.

|| Present address: Dept. of Chemistry and Biochemistry, 601 University Dr., Southwest Texas State University, San Marcos, TX 78666.

** To whom correspondence should be addressed: Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8331; Fax: 81-6-6877-3584; E-mail: asugino@biken.osaka-u.ac.jp.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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