Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli

Gawel, Damian; Fijałkowska, Iwona J.; Jonczyk, Piotr; Schaaper, Roel M.

doi:10.1016/j.mrfmmm.2013.11.003

Cited by 16 publications

(13 citation statements)

References 36 publications

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“…However, we have no such explanation for the twofold bias for A:T transitions to occur when A is on the LGST, although we speculated that DNA polymerase was more likely to insert a C opposite A when the A was templating lagging-strand synthesis (Lee et al 2012). In contrast, other studies have indicated that leading strand DNA synthesis is more error-prone than lagging strand synthesis (Gawel et al 2014). The results presented here with a strain defective for both MMR and Ndk support our original hypothesis, suggesting that lagging-strand synthesis is more error prone, at least when C is misinserted opposite template A or T. However, the mutational spectrum seen in the absence of MMR is the result of replication errors that are not corrected by the proofreader.…”

Section: Discussionmentioning

confidence: 63%

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair DefectiveEscherichia coli

et al. 2018

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Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defective strains yielded ≈30,000 base-pair substitutions (BPSs), revealing mutational patterns across the entire chromosome. The BPS spectrum was dominated by A:T to G:C transitions, which occurred predominantly at the center base of 5'NC3'+5'GN3' triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for BPSs, and the rate at which these occurred increased with run length. Comparison with ≈2000 BPSs accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an double mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone, or less well corrected by proofreading, than was leading strand synthesis.

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Section: Discussionmentioning

confidence: 63%

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair DefectiveEscherichia coli

et al. 2018

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“…It is well documented that dNTP pool imbalances affect the rate and spectrum of spontaneous mutation, presumably by driving preferential incorporation of the nucleotide(s) in excess (59,63). 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).…”

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

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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“…Mutations in both ndk and dcd were used in a recent study by Gawel et al (9), who asked whether DNA replication fidelity in E. coli is higher on the leading or lagging strand. Even though in bacteria both strands are replicated by the same polymerase, previous studies had suggested that fidelity is higher for lagging‐strand replication, a seemingly counterintuitive finding because of the more complex process of discontinuous DNA synthesis on the lagging strand.…”

Section: Mutagenic Mechanisms Inferred From Nucleotide Pool Variationsmentioning

confidence: 99%

Deoxyribonucleotides as genetic and metabolic regulators

Mathews

2014

FASEB j.

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For >35 yr, we have known that the accuracy of DNA replication is controlled in large part by the relative concentrations of the 4 canonical deoxyribonucleoside 5'-triphosphates (dNTPs) at the replisome. Since this field was last reviewed, ∼8 yr ago, there has been increased understanding of the mutagenic pathways as they occur in living cells. At the same time, aspects of deoxyribonucleotide metabolism have been shown to be critically involved in processes as diverse as cell cycle control, protooncogene expression, cellular defense against HIV infection, replication rate control, telomere length control, and mitochondrial function. Evidence supports a relationship between dNTP pools and microsatellite repeat instability. Relationships between dNTP synthesis and breakdown in controlling steady-state pools have become better defined. In addition, new experimental approaches have allowed definitive analysis of mutational pathways induced by dNTP pool abnormalities, both in Escherichia coli and in yeast. Finally, ribonucleoside triphosphate (rNTP) pools have been shown to be critical determinants of DNA replication fidelity. These developments are discussed in this review article.

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Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli

Cited by 16 publications

References 36 publications

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair DefectiveEscherichia coli

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair DefectiveEscherichia coli

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

Deoxyribonucleotides as genetic and metabolic regulators

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