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 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.
The Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Kikuo
K
Shimizu
Keiji
K
Hashimoto
Jake M
JM
Kirchner
Wataru
W
Nakai
Hiroko
H
Nishikawa
Michael A
MA
Resnick
Akio
A
Sugino
D001483:Base Sequence; D004257:DNA Polymerase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013698:Templates, Genetic
D017931:DNA Primers; D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D004257:DNA Polymerase II; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V