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

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.

Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand

“…Most previous experimental studies confirm the classical model, including experiments with mutant polymerases (Pursell et al 2007;Kunkel and Burgers 2008;Nick McElhinny et al 2008;Johnson et al 2015) or incorporation of ribonucleotides Clausen et al 2015;Johnson et al 2015), where specific mutations were observed on the corresponding strands; experiments investigating the association of proteins with leading and lagging strands of DNA replication forks (Yu et al 2014); and biochemical experiments of assembly and stabilization of replication complexes (Georgescu et al , 2015Langston et al 2014). Moreover, recent evidence suggests that Pol epsilon does not proofread errors made by Pol delta (Flood et al 2015). This model (Johnson et al 2015) also contradicts our data, as it does not predict the opposite strand biases we observe in cancers with mutated Pol epsilon and Pol delta (Fig.…”

Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae

“…Given evidence that the exonuclease activity of Pol δ, but not that of Pol ε, proofreads errors made by Pol α[12], and evidence that Pol δcan also proofread errors made by Pol ε [13], the higher mutation rate in the pol3-exo - msh6 Δ strain could be due to loss Pol δ proofreading of errors made by any of the three replicases, whereas loss Pol ε may only proofread its own errors. The different mutation rates in the pol3-exo - msh6 Δ and pol2-exo - msh6 Δ strains could also be related to differences in activation of the S phase checkpoint in proofreading-deficient strains (see [26] and references therein).…”

“…Thus it is possible that in the absence of only one of the two proofreading activities intrinsic to Pol ε and Pol δ, the exonuclease that remains intact in one replicase can proofread mismatches generated by the other major, but proofreading-deficient replicase [2,12]. Indeed, a recent study [13] provides evidence that Pol δ proofreads errors made by Pol ε. Theoretically, extrinsic proofreading may also be catalyzed by other 3′-exonucleases (e.g., see [52]). To the extent that extrinsic proofreading may occur during replication in yeast, this implies that (i) the actual base selectivity of the replicases in vivo could be substantially lower than calculated here, and therefore more in line with the estimates from studies in vitro , and (ii) the base selectivity calculated here for lagging strand replication may be a mixture of the base selectivity of Pol α plus Pol ε, because both polymerases contribute to the mature lagging strand (see [21,53], and more recently [5,8]).…”

“…Moreover, there is evidence to suggest that the exonuclease activity of Pol δ, but not that of Pol ε, likely proofreads errors made by Pol α during lagging strand replication [12], and more recent evidence that Pol δ can proofread errors made by Pol ε [13]. Not only is proofreading more complicated in yeast as compared to E. coli , but so too is eukaryotic MMR more complicated than in E. coli .…”

Replicative DNA polymerase defects in human cancers: Consequences, mechanisms, and implications for therapy

“…Polε exonuclease deficiency results in a very small increase in the mutation rate in both yeast and human cells [8,83,84], even though the fidelity of purified Polε in vitro is strongly affected by the inactivation of proofreading [85,86]. It has been suggested that the majority of Polε errors are corrected in cells by extrinsic mechanisms, for example, by the exonuclease activity of Polδ [2,87]. On the other hand, many Polε exonuclease domain mutations found in cancers, and particularly P286R, were predicted to affect DNA binding [23,24,63].…”