Abstract
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.
Highlights
THE JOURNAL OF BIOLOGICAL CHEMISTRYVol 277, No 40, Issue of October 4, pp. 37422–37429, 2002 Printed in U.S.A. Fidelity of DNA Polymerase ⑀ Holoenzyme from Budding Yeast Saccharomyces cerevisiae*
DNA polymerases ␦ and ⑀ are the major replicative polymerases and possess 3-5 proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae
The yeast Saccharomyces cerevisiae has three DNA polymerases, which are required for cell growth, chromosomal DNA replication [1], and DNA double-strand break repair [2]. pol ␣ has four subunits (Pol1 (Cdc17), Pol10, Pri1, and Pri2) and is involved primarily in the initiation of DNA replication and priming of Okazaki fragments. pol ␦ and pol ⑀ are required during synthesis of the leading and lagging DNA strands at the replication fork; they bind at/or near replication origins and move along DNA with the replication fork [3, 4]
Summary
Vol 277, No 40, Issue of October 4, pp. 37422–37429, 2002 Printed in U.S.A. Fidelity of DNA Polymerase ⑀ Holoenzyme from Budding Yeast Saccharomyces cerevisiae*. Pol is the catalytic subunit of pol ⑀ and is encoded by the POL2 gene [15], which is essential for cell growth and required for chromosomal DNA replication [16]. The DPB3 and DPB4 genes encode the third and fourth subunits of pol ⑀, respectively [18, 19] These genes are not essential for cell growth, they play an important role in stabilizing the subunit structure of pol ⑀ and in interactions between pol ⑀ and other DNA replication proteins [19]. A pol deletion mutant is temperature-sensitive, exhibits severe defects in chromosomal DNA replication at the permissive temperature, and undergoes premature senescence [25] This suggests that other DNA polymerases can substitute for the polymerase function of pol ⑀ but that another unspecified function of pol ⑀ is essential in yeast. This study measures the fidelity of wild-type, pol , pol2C1089Y, and pol2C1089Y pol enzymes using the synthetic 30-mer primer/100-mer template employed in previous studies [29, 32]
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