DNA Polymerase ε: A Polymerase of Unusual Size (and Complexity)

Pursell, Zachary F.; Kunkel, Thomas A.

doi:10.1016/s0079-6603(08)00004-4

Cited by 76 publications

(79 citation statements)

References 215 publications

(216 reference statements)

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“…plays a substantial role in genome replication (Pursell and Kunkel 2008). Our data also indicate that loss of genetic information on either DNA strand [Pol e errors on leadingstrands and Pol d errors on lagging-strands (Pursell et al 2007;Nick McElhinny et al 2008)] is sufficient to drive extinction of a population of cells.…”

Section: Pol E Errors and Lethal Mutagenesismentioning

confidence: 55%

“…Three additional regions of homology (C-1, C-2, and C-3) are framed in black (Huang et al 1999). Amino-acid conservation is indicated using the following color scheme: red, residues conserved in all four sequences; yellow, residues conserved in three sequences; gray, similar amino acids in at least three sequences.Antimutator Variants of DNA Polymerase e 759 plays a substantial role in genome replication (Pursell and Kunkel 2008). Our data also indicate that loss of genetic information on either DNA strand [Pol e errors on leadingstrands and Pol d errors on lagging-strands (Pursell et al 2007;Nick McElhinny et al 2008)] is sufficient to drive extinction of a population of cells.…”

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confidence: 52%

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

2013

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DNA polymerases (Pols) e and d perform the bulk of yeast leading-and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10-to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol d proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol e proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2D, mlh1D, msh3D msh6D, or pms1D mlh3D) but not with partial MMR loss (msh3D, msh6D, pms1D, or mlh3D), indicating that high levels of unrepaired Pol e errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2D eex mutants encoded second-site changes in Pol e that reduced the pol2-4 mutator phenotype between 3-and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol e and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading-and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol e gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.O RGANISMS must accurately duplicate their genomes to avoid loss of long-term fitness. Consequently, cells employ high-fidelity DNA polymerases (Pols) equipped with proofreading exonucleases to replicate their DNA (reviewed in McCulloch and Kunkel 2008;Reha-Krantz 2010). Mismatch repair (MMR) further ensures the integrity of genetic information by targeting mismatches for excision from newly replicated DNA (reviewed in Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008). These polymerase error-correcting mechanisms, together with DNA damage repair (Friedberg et al. 2006), maintain the genome with less than one mutation per 10 9 nucleotides per cell division (Drake et al. 1998). Defects in proofreading or MMR result in mutator phenotypes characterized by increased rates of spontaneous mutation (Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008;McCulloch and Kunkel 2008; RehaKrantz 2010).Mutator phenotypes can be an important source of genetic diversity, which facilitates adaptation to environmental change. In bacterial and yeast populations, unstable environments favor mutator strains that readily acquire adaptive mutations (Chao and Cox 1983;Mao et al. 1997;Sniegowski et al. 1997...

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Section: Pol E Errors and Lethal Mutagenesismentioning

confidence: 55%

mentioning

confidence: 52%

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

2013

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“…Earlier skepticism that Pol d was simply Pol a contaminated with a 3 0 -5 0 exonuclease activity was dispelled by the demonstration that both the polymerase and exonuclease activities were associated with the p125 catalytic subunit [Lee et al, 1980;Lee et al, 1984]. A second form of ''Pol d'' with a larger catalytic subunit was discovered and is now known as Pol e [Syvaoja et al, 1990;Pospiech and Syvaoja, 2003;Pursell and Kunkel, 2008]. Molecular cloning of p125 showed that the catalytic core was highly conserved in evolution from T4 bacteriophage to human Hao et al, 1992;Yang et al, 1992], and Pol d is now classified as a member of the B family polymerases.…”

Section: Introductionmentioning

confidence: 99%

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Lee

Zhang

Lin

et al. 2012

Environ and Mol Mutagen

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The p12 subunit of polymerase delta (Pol d) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol d4 holoenzyme to the heterotrimer, Pol d3. We review studies that establish that Pol d3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol d3 exhibits properties of an ''antimutator'' polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol d carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol d to sites of DNA damage show that Pol d3 is the primary form of Pol d associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol d engaged in DNA repair. We propose a model for the switching of Pol d with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol d3. Because of the critical role of Pol d activity in DNA replication and repair, the formation of Pol d3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Environ. Mol. Mutagen. 53:683-698, 2012. V V C 2012 Wiley Periodicals, Inc.

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“…Possibilities include signaling for mismatch repair, nucleosome loading behind the replication fork, chromatin remodeling, and gene silencing. The presence of a single ribonucleotide in DNA has been shown to reduce nucleosome formation (42 79), and several studies (reviewed in (43)) have shown that Pol ε is involved in gene silencing and one of its noncatalytic subunits is involved in chromatin remodeling. Moreover, mutations in S. pombe cdc22, and tds1 genes, encoding the large subunit of ribonucleotide reductase and a putative thymidylate synthase, respectively, cause spreading of silencing across heterochromatic barriers in the mating-type switching region (44).…”

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confidence: 99%

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

McElhinny

Watts

Kumar

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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582

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Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36-to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols α, δ, and ε, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...

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DNA Polymerase ε: A Polymerase of Unusual Size (and Complexity)

Cited by 76 publications

References 215 publications

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

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

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

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