Inefficient proofreading and biased error rates during inaccurate DNA synthesis by a mutant derivative of Saccharomyces cerevisiae DNA polymerase

Stephanie A.Nick McElhinny, Carrie M. Stith, Peter M.J. Burgers, Thomas A. Kunkel

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

DNA polymerase δ (pol δ) is a high fidelity eukaryotic enzyme that participates in DNA repair and is essential for DNA replication. Toward the goal of dissecting its multiple biological functions, here we describe the biochemical properties of Saccharomyces cerevisiae pol δ with a methionine replacing conserved leucine 612 at the polymerase active site. Compared with wild type pol δ, L612M pol δhas normal processivity and slightly higher polymerase specific activity. L612M pol δ also has normal 3′ exonuclease activity, yet it is impaired in partitioning mismatches to the exonuclease active site, thereby reducing DNA synthesis fidelity. Error rates in vitro for L612M pol δ are elevated for both base substitutions and single base deletions but in a highly biased manner. For each of the six possible pairs of reciprocal mismatches that could arise during replication of complementary DNA strands to account for any particular base substitution in vivo (e.g. T-dGMP or A-dCMP for T to C transitions), L612M pol δ error rates are substantially higher for one mismatch than the other. These results provide a biochemical explanation for our observation, which confirms earlier genetic studies, that a haploid pol3-L612M S. cerevisiae strain has an elevated spontaneous mutation rate that is likely due to reduced replication fidelity in vivo.

Original languageEnglish
Pages (from-to)2324-2332
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number4
DOIs
StatePublished - Jan 26 2007

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