Checkpoint-Dependent Activation of Mutagenic Repair in Saccharomyces cerevisiae pol3-01 Mutants

Datta, Abhijit; Schmeits, James L.; Amin, Neelam S.; Lau, Patrick J.; Myung, Kyungjae; Kolodner, Richard D.

doi:10.1016/s1097-2765(00)00058-7

Cited by 88 publications

(81 citation statements)

References 65 publications

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“…5 could be triggered by mutations other than pol3-R696W, and whether it can operate in human cells. In yeast, the requirement of Dun1 for the mutator effect has been observed previously for two other Polδ variants (33,73). These findings suggest that high sensitivity of replicative DNA polymerase mutants to fluctuation of intracellular dNTP levels could be a general phenomenon.…”

Section: Discussionsupporting

confidence: 75%

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

Mertz

Sharma²,

Chabes

et al. 2015

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

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Defects in DNA polymerases δ (Polδ) and e (Pole) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.DNA polymerase δ | colon cancer | dNTP pools | mutagenesis | DNA replication fidelity W hen functioning properly, DNA replication is phenomenally accurate, making ∼10 −10 mutations per base pair during each replication cycle (1). In respect to human biology, it means that, on average, less than one mutation occurs each time the human genome is replicated. This amazing exactitude is contingent upon the serial action of DNA polymerase selectivity, exonucleolytic proofreading, and DNA mismatch repair (MMR). MMR defects increase spontaneous mutagenesis in numerous model systems (2) and give rise to cancer in mice (3). In humans, inherited mutations in MMR genes predispose to colorectal cancer (CRC) in Lynch syndrome (4, 5). Additionally, MMR genes are inactivated via hypermethylation in ∼15% of sporadic CRC, endometrial cancer (EC), and gastric cancer (6).Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7-14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporad...

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Section: Discussionsupporting

confidence: 75%

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

Mertz

Sharma²,

Chabes

et al. 2015

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

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“…The pol2-4 and pol2-M644G alleles increase mutation rates threefold and 10-fold, respectively ( Fig. 2A (5,19,21,45). Mutation rates of pol2-4 dun1Δ cells were not statistically different from mutation rates of WT or dun1Δ cells, whereas pol2-M644G dun1Δ cells failed to form visible colonies upon shuffling ( Fig.…”

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

confidence: 92%

“…Previous studies observed that the mutator phenotype of proofreading-deficient Pol δ (encoded by the pol3-01 allele) partially depends on the Dun1 effector kinase (20,21) (Fig. 1), which lies directly downstream of the mitosis entry checkpoint 1 (Mec1) [mammalian ataxia telangiectasia and Rad3-related protein (ATR)] and Rad53 [mammalian checkpoint kinase (Chk)1] kinases in the S-phase checkpoint pathway (22,23) (Fig.…”

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confidence: 99%

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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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“…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).…”

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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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Checkpoint-Dependent Activation of Mutagenic Repair in Saccharomyces cerevisiae pol3-01 Mutants

Cited by 88 publications

References 65 publications

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

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

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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