Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli

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

Section: Discussionsupporting

confidence: 54%

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

Williams

Marjavaara

Knowels

et al. 2015

“…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). Less recognized are the differences in the mutational specificity of DNA polymerases that could result from using a physiological concentration of each dNTP in the in vitro reactions as opposed to commonly used equimolar dNTPs.…”

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

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...

“…Failure to increase dNTP levels at the G 1 -to-S transition of the cell cycle is a lethal event at cellular level (2,3). Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4)(5)(6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7)(8)(9).…”

mentioning

confidence: 99%

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Zhang

Yang

et al. 2006

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (␣, oligomeric state unknown) and a small subunit R2 (␤ 2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe) 2-Y⅐] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (␤␤ ), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ␤␤ is redistributed from the nucleus to the cytoplasm in a checkpointdependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ␤␤ nuclear localization: the importin ␤ homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ␤␤ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ␤␤ . Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ␤␤ by Kap122 or as an anchor to retain ␤␤ in the nucleus. DNA-damage checkpoint ͉ subcellular redistributionT he levels and relative ratios of dNTP pools are important for high-fidelity DNA replication and repair (1). Failure to increase dNTP levels at the G 1 -to-S transition of the cell cycle is a lethal event at cellular level (2, 3). Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4-6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7-9). Eukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to ensure proper dNTP pool sizes during the normal cell-cycle progression and in response to genotoxic stress (3, 10-14). A major target of such checkpoint regulation is ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate, an essential step in de novo biosynthesis of dNTPs (15).Class I RNRs were identified originally in Escherichia coli and are conserved from yeast to mammal (16). The mechanisms of enzymatic catalysis (17) and allosteric regulation (18, 19) have been studied extensively in E. coli and, more recently, in mice (20, 21). The archetype RNR holoenzyme consists of a large subunit R1 (␣, whose oligomeric state in eukaryotes is not completely understood) (22) and a homodimeric small subunit (␤ 2 ) (20). The eukaryotic R1 contains the catalytic site, an effector site that controls substrate specificity, an activity site that controls turnover, and a weak ATP-binding site that cont...