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Papers In Press, published online ahead of print July 17, 2002
J. Biol. Chem, 10.1074/jbc.M204476200
Submitted on May 7, 2002
Revised on July 8, 2002
Accepted on July 16, 2002

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

Kikuo Shimizu, Keiji Hashimoto, Jake M Kirchner, Wataru Nakai, Hiroko Nishikawa, Michael A Resnick, and Akio Sugino

Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871

Corresponding Author: asugino{at}biken.osaka-u.ac.jp

DNA polymerases {d }and {e} (pol {d} and {e}) are the major replicative polymerases and possess 3’-5’ proofreading exonuclease activities which correct errors that arise during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of holoenzyme of wild-type pol {e}, 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 {e} was 0.46 x 10-5 for G:G mispairs, 015 x 10-5 for T:G mispairs, and less than 0.01 x 10-5 for A:G mispairs. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol {e}, however, G:G and T:G misincorporation rates increased 40- and 78-fold, respectively. The pol2C1089Y pol {e} mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold , respectively, while A:G misincorporation did not differ from 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 {e} is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.


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