Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

Youri I Pavlov,Satoko Maki,Hisaji Maki,Thomas A Kunkel

doi:10.1186/1741-7007-2-11

Abstract

BackgroundDNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect.ResultsPurified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo.ConclusionsOur data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.

Highlights

DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast
Pol α is not very processive and lacks an intrinsic proofreading exonuclease. It has a tightly associated activity for the synthesis of RNA primers at replication origins and on the lagging DNA strand. Pol α extends these RNA primers by synthesizing short stretches of DNA, and a switch occurs to processive synthesis by Pol ε and/or Pol δ
Investigation of the mechanism of the mutator phenotype of the yeast Y831A DNA Pol ε mutant began with the observation that, for an equivalent amount of polymerization activity, the purified Y831A enzyme has an increased ability to convert dNTPs to dNMPs relative to wild-type Pol ε (Figure 2)

Summary

Introduction

DNA polymerase ε (Pol ε) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. Multiple DNA polymerases are thought to be present at the eukaryotic replication fork [1,2,3,4] Some of their functions could be unique while others could be overlapping. Pol α is not very processive and lacks an intrinsic proofreading exonuclease It has a tightly associated activity for the synthesis of RNA primers at replication origins and on the lagging DNA strand. Pol α extends these RNA primers by synthesizing short stretches of DNA, and a switch occurs to processive synthesis by Pol ε and/or Pol δ

Methods

Results

Discussion

Conclusion