A novel function of DNA polymerase ζ regulated by PCNA

Northam, Matthew R.; Garg, Parie; Baitin, Dmitry; Burgers, Peter M.; Shcherbakova, Polina V.

doi:10.1038/sj.emboj.7601320

Cited by 96 publications

(203 citation statements)

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“…We have shown previously that many mutations in replicative DNA polymerases can lead to an increased spontaneous mutation rate indirectly through the recruitment of the error-prone Polζ (21). To determine if any of the spontaneous mutagenesis in pol3-R696W/pol3-R696W (1) and (2) were constructed using PSD93 and E134×1B-D770 strains, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…The rate of forward mutation to Can r was measured by fluctuation analysis as described previously (41). Immunoblot analysis of whole-cell extracts was performed as described (21) with rabbit polyclonal antiserum to yeast Polδ (42) provided by Peter Burgers (St. Louis, MO). Polδ and Polδ-R696W were overproduced and purified as previously described (43) with minor modification (see SI Text).…”

Section: Methodsmentioning

confidence: 99%

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A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

Daee

Mertz

Shcherbakova

2009

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

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Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino-acid changes in Polδ or Polε. In addition, Polδ variants were found in colon-cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancerassociated Polδ mutations and four polymorphisms affecting wellconserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

Daee

Mertz

Shcherbakova

2009

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

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“…Error-prone synthesis by Pol ζ and Pol η allows bypass of replication-blocking DNA lesions (46), and several mutator polymerases are known to create mutations in a Pol ζ-dependent manner (47)(48)(49)(50)(51)(52). To test whether Pol ζ or Pol η lies downstream of Dun1 and contributes to the pol2 mutator phenotypes, we engineered pol2-4 and pol2-M644G strains lacking Pol ζ (rev3Δ) or Pol η (rad30Δ).…”

Section: Dun1 Is Required For the Mutator Phenotype Or Viability Of Pol2mentioning

confidence: 99%

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

Williams

Marjavaara

Knowels

et al. 2015

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

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Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) e mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol e depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol e proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol e base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol e mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.DNA replication and repair | polymerase fidelity | cancer | lethal mutagenesis

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“…Studies of T4 antimutators show that enhanced discrimination against some errors is commonly accompanied by weakened discrimination against others (Drake 1993). Polymerase variants with increased fidelity often have reduced overall activity (Loh et al 2007), which can improve accuracy (Clayton et al 1979;Kunkel et al 1994;Joyce and Benkovic 2004;Johnson 2010;Herr et al 2011b), but will also increase error rates for some mutation types (Kunkel et al 1994) and may trigger mutagenic replication processes in the cell (Northam et al 2006;Aksenova et al 2010;Northam et al 2010). Additionally, T850M and K966Q would increase mutagenesis if they hamper partitioning of nascent errors to the exonuclease active site (Donlin et al 1991;Reha-Krantz 2010).…”

Section: Escape From Pol E Error-induced Extinctionmentioning

confidence: 99%

“…Serial dilutions were plated on FOAcontaining media to select for loss of complementing POL2-or POL3-URA3 plasmids and reveal the synthetic phenotype. POL and MMR alleles are indicated at left and above the corresponding panels, respectively.Antimutator Variants of DNA Polymerase e 7552003; Northam et al 2006;Aksenova et al 2010;Northam et al 2010). This dependency may reflect a role for Pol z in rescuing stalled DNA replication forks (Northam et al 2010) and may relate to Pol z's ability to efficiently extend primers with 39-terminal mismatches (Johnson et al 2000;Guo et al 2001;Haracska et al 2001;Simhadri et al 2002).…”

mentioning

confidence: 99%

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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A novel function of DNA polymerase ζ regulated by PCNA

Cited by 96 publications

References 54 publications

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants

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

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