Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

• Type of Material: Article
• Authors: Carrie L. Flood, Emory University; Gina P. Rodriguez, Emory University; Gaobin Bao, Emory University; Arthur H. Shockley, Emory University; Yoke Kow, Emory University; Gray Crouse, Emory University
• Language: English
• Date: 2015-03
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2015 Flood et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pgen.1005049
• Title of Journal or Parent Work: PLoS Genetics
• ISSN: 1553-7390
• Volume: 11
• Issue: 3
• Start Page: e1005049
• End Page: e1005049
• Grant/Funding Information: This work was supported by the This work was supported by the National Institutes of Health [R01 GM80754 to GFC and P01 ES011163 to GFC and YWK].
• Research Categories: Biology, Molecular; Biology, Genetics

Abstract

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3'-terminal 8 oxoG, unlike a 3'-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.