Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-10, Vol.277 (40), p.37422
Main Authors: Shimizu, Kikuo, Hashimoto, Keiji, Kirchner, Jake M, Nakai, Wataru, Nishikawa, Hiroko, Resnick, Michael A, Sugino, Akio
Format: Article
Language:English
Subjects:
Base Sequence
Deoxyribonucleotides - metabolism
DNA Polymerase II - chemistry
DNA Polymerase II - genetics
DNA Polymerase II - metabolism
DNA Primers
DNA Replication
Exodeoxyribonuclease V
Exodeoxyribonucleases - genetics
Gene Deletion
Kinetics
Molecular Sequence Data
Mutagenesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Templates, Genetic
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Summary: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 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M204476200