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

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...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Mertz

Sharma²,

Chabes

et al. 2015

“…They map in the dnaE gene encoding the α (polymerase) subunit of PolIII, and which have been broadly characterized as antimutators (21,24). Error catastrophe has also been described in the yeast S. cerevisiae, where certain proofreading-deficient variants of DNA polymerase delta or epsilon are incompatible with a complete deficiency of DNA mismatch repair (25,26). Suppressors of this incompatibility (termed eex) can also be obtained, which reduce the mutation rate sufficiently to permit life (25,26).…”

Section: E Coli Mutator Mutants At the Rnr Specificity Site: Exceptimentioning

confidence: 99%

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

Ahluwalia

Schaaper

2013

The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.T he enzyme ribonucleotide reductase (RNR) is a critical cellular factor for the synthesis and control of the deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA replication and DNA repair (1). Specifically, RNR is responsible for the reduction of the ribonucleotides to the corresponding deoxynucleotides. In many well-studied cell systems, like the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, or mammalian cells, this reduction takes place at the level of the nucleoside diphosphates (NDP → dNDP), and each of four separate NDPs (ADP, GDP, CDP, and UDP) can serve as RNR substrate (1, 2). Following reduction, the dNDPs are converted to the corresponding dNTPs by nucleoside diphosphate kinase (NDK) (3). The DNA precursor dTTP is not generated directly through this pathway; instead, it is produced from dCTP via dUTP (4).RNRs have been subdivided into several classes, depending on the type of radical used in the catalytic reaction (1, 5-7). The class Ia RNR, as present in E. coli, yeast, and mammalian cells, employs a tyrosyl radical. Structurally, these RNRs are tetramers composed of a dimer of a large subunit (R1) and a dimer of a small subunit (R2). The large subunits contain the catalytic site and two allosteric regulatory sites (termed specificity site and activity site), whereas the small subunit contains the essential tyrosyl radical. The activity site is located at the N terminus of the R1 subunit and functions as an "on-off" switch: ATP binding leads to an active enzyme, whereas dATP binding inhibits the enzyme. Hence, by monitoring the ATP/dATP ratio, the enzyme aims to ensure an overall dNTP level that is presumably optimal for DNA replication (1, 8).The R1 specificity site, is a binding site for dATP, ATP,...

“…Given the high conservation of DNA replication machinery, the yeast Saccharomyces cerevisiae represents an ideal system with which to identify genetic pathways that influence mutator phenotypes (18)(19)(20). The pol2-4 allele, encoding proofreadingdeficient Pol e, is lethal in strains lacking all MMR activity [e.g., deletion of MutS homologue (MSH) 2 (msh2Δ)] (19).…”

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confidence: 99%

“…The pol2-4 allele, encoding proofreadingdeficient Pol e, is lethal in strains lacking all MMR activity [e.g., deletion of MutS homologue (MSH) 2 (msh2Δ)] (19). This "errorinduced extinction" is surmounted by "antimutator" mutations affecting the Pol e catalytic subunit, as well as by unidentified mutations elsewhere in the genome (19). Mutants harboring these Significance An increased rate of mutation, or "mutator phenotype," generates genetic diversity that can accelerate cancer progression or confer resistance to chemotherapy drugs.…”

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confidence: 99%

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

Williams

Marjavaara

Knowels

et al. 2015

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