Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Abstract

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ⑀ (Pol ⑀) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ⑀ synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of <2 ؋ 10 ؊5 and <5 ؋ 10 ؊7 , respectively. Pol ⑀ lacking 3 3 5 exonuclease activity is less accurate to a degree suggesting that wild type Pol ⑀ proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ⑀ is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T⅐dTTP, T⅐dCTP, and C⅐dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ⑀ differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ⑀ in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ⑀ proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ⑀ polymerase activity in DNA replication.Replication of chromosomes in eukaryotes is believed to require three DNA polymerases, Pol ␣, 1 Pol ␦, and Pol ⑀ (for reviews, see Refs. 1-3). Pol ␣ has an associated DNA primase activity and synthesizes short RNA-DNA primers to initiate replication at origins and start Okazaki fragments on the lagging DNA strand. The roles of Pol ␦ and Pol ⑀ in chromosomal replication are not clearly defined. Evidence for essential roles of Pol ␦ and Pol ⑀ in replication comes from genetic studies in Saccharomyces cerevisiae. Mutational inactivation of catalytic subunits and some of the additional subunits of both polymerases is lethal. Strains carrying temperature-sensitive mutations in POL3 gene (encoding the catalytic subunit of Pol ␦), POL2, or DPB2 genes (encoding subunits of Pol ⑀) arrest in S phase of the cell cycle with a terminal morphology characteristic of a DNA replication defect upon shift to non-permissive temperature (4 -6). Incorporation of labeled precursors into DNA stops in these mutants at non-permissive temperature (4, 5, 7). The POL3, POL2, and DPB2 genes, as well as the DPB3 gene encoding the third subunit of Pol ⑀, are expressed periodically in the cell cycle with a peak in G 1 to S phase transition (4, 5, 8), which is characteristic of genes encoding DNA replication proteins. Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9, 10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).To accommodate roles for Pol ␦ and Pol ⑀ at a replication fork, it was proposed...

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

“…It is well documented that dNTP pool imbalances affect the rate and spectrum of spontaneous mutation, presumably by driving preferential incorporation of the nucleotide(s) in excess (59,63). The potential effects of proportional increases or decreases in dNTP levels on DNA polymerase fidelity could also be predicted from kinetic considerations and have been demonstrated in a few biochemical studies (64)(65)(66). Significant stimulation of mutagenesis in E. coli by proportional dNTP increases has been reported (67,68).…”

“…Transformation of E. coli with the gap-filling reactions, scoring of mutant plaques, and ssDNA isolation from purified plaques were as described by Bebenek and Kunkel (43). The lacZ gene from mutant M13mp2 plaques was sequenced, and error rates were calculated as previously described (66).…”

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

“…Tran et al (1999) show that loss of Msh2-Msh3 (msh3D) further increases the mutation rate of pol2-4 msh6D cells about threefold, while we find that msh3D is lethal in pol2-4 msh6D cells and therefore must increase mutations $10-fold. A 10-fold increase corresponds to $3 · 10 26 mutations/base pair, which is substantially lower than the error rate of proofreading-deficient Pol e in vitro [$3 · 10 24 errors/base pair (Shcherbakova et al 2003)]. Furthermore, the spectra of mutations observed in vivo and in vitro are quite different ( Figure 6 and Table 2) (Shcherbakova et al 2003).…”

“…Defects in Pol e or Pol d proofreading increase the spontaneous mutation rate in a manner consistent with major roles for these polymerases in leadingand lagging-strand synthesis (Morrison et al 1991;Simon et al 1991;Morrison and Sugino 1994;Shcherbakova et al 1996;Tran et al 1999;Karthikeyan et al 2000;Greene and Jinks-Robertson 2001). Interestingly, the Pol d proofreading defect generates a mutator phenotype 5-to 30-fold greater than that observed in Pol e proofreading-deficient strains (Morrison and Sugino 1994;Shcherbakova et al 1996;Tran et al 1999;Datta et al 2000;Karthikeyan et al 2000;Greene and Jinks-Robertson 2001;Pavlov et al 2004), and the spectra of spontaneous mutations that arise in Pol e and Pol d proofreading-deficient strains differ, which may reflect distinct error specificities of the polymerases as well as strand-specific effects (Morrison and Sugino 1994;Karthikeyan et al 2000;Pavlov et al 2002Pavlov et al , 2003Shcherbakova et al 2003;Fortune et al 2005). Mouse cells with defects in Pol e or Pol d proofreading also exhibit increased mutation rates (Goldsby et al 2002;Albertson et al 2009), and, consistent with distinct roles in DNA replication, the types of tumors that develop in Pol e and Pol d proofreading-deficient mice differ markedly (Albertson et al 2009).…”

“…Both polymerases are accurate and possess intrinsic proofreading exonucleases that edit mispaired primer termini during polymerization (Morrison et al 1991;Simon et al 1991;Shimizu et al 2002;Shcherbakova et al 2003;Fortune et al 2005). Defects in Pol e or Pol d proofreading increase the spontaneous mutation rate in a manner consistent with major roles for these polymerases in leadingand lagging-strand synthesis (Morrison et al 1991;Simon et al 1991;Morrison and Sugino 1994;Shcherbakova et al 1996;Tran et al 1999;Karthikeyan et al 2000;Greene and Jinks-Robertson 2001).…”