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

Purified 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.Our 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.Multiple DNA polymerases are thought to be present at the eukaryotic replication fork [1-4]. Some of their functions could be unique while others could be overlapping. Different polymerases may compete for certain DNA substrates and several polymerases may sometimes act in concert [5-9]. Under normal circumstances, chromosomal replication requires at least three DNA polymerases, Pol α, Pol ε and Pol δ. All of these polymerases are multi-subunit complexes [1,4] and all subunits are required for their proper function (see recent papers [10-12], and references therein). 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 then a switch occurs to processive synthesis by Pol ε and/or Pol δ.T