Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype

Davidson, Marta B.; Katou, Yuki; Keszthelyi, Andrea; Sing, Tina L.; Xia, Tian; Ou, Jiongwen; Vaisica, Jessica A.; Thevakumaran, Neroshan; Marjavaara, Lisette; Myers, Chad L.; Chabes, Andrei; Shirahige, Katsuhiko; Brown, Grant W.

doi:10.1038/emboj.2011.485

Cited by 97 publications

(95 citation statements)

References 104 publications

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“…This dNTP pool increase is important for survival and mutagenesis after exposure to DNA damaging agents (47,48). Dun1 has also been reported to promote expansion of dNTP pools in response to mutations that impair DNA replication (49). Considering these findings and the suppression of mutagenesis in pol3-R696W strains by the DUN1 deletion, we hypothesized that the mutator effect of pol3-R696W requires a Dun1-dependent increase in dNTP pools.…”

Section: Resultsmentioning

confidence: 97%

“…In comparison, the full activation of the checkpoint by DNA damaging agents leads to a sixfold to eightfold increase in dNTP levels (47). Other studies similarly reported that Rad53 phosphorylation is often not detectable in cells with chronic or low checkpoint activation even though effects of downstream signaling are apparent (49,55,56). Due to the signal amplification by the kinase cascade, the levels of the downstream targets, such as Sml1 or the RNR subunits (46, 57), are much more sensitive indicators of the checkpoint activation.…”

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confidence: 99%

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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: Resultsmentioning

confidence: 97%

mentioning

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.

Self Cite

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“…Others have observed that RNR1 expression is independent of Dun1 activity and induced up to fivefold during checkpoint activation (28,37), whereas RNR3 induction is induced up to 100-fold (28). Although initially thought to be solely Dun1-dependent (31), RNR3 induction can also occur by a Dun1-independent pathway (30,57,58). To investigate the checkpoint response to Pol e mutations, we measured RNR1 and RNR3 transcript levels in POL2, pol2-4, and pol2-M644G strains.…”

Section: Pol2-4 and Pol2-m644g Exhibit Divergent Effects On Cell Cyclementioning

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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“…However, the trade-off for this damage repair response is a TLS-dependent increase in mutation rates. Recently, RNR induction and expansion of dNTP pools were recorded in tsa1D and other genome instability mutants deficient in various seemingly unrelated processes (repli-cation fork progression, recombinational repair, and sister chromatid cohesion) (Davidson et al 2012;Poli et al 2012). By analyzing these data, it appears as if a tsa1D mutant displays higher mutation rates than expected from the dNTP pools alone, suggesting that Tsa1 deficiency results in additional problems related to mutation protection.…”

Section: Links Between Genome Instability Cancer and Prx Activitymentioning

confidence: 99%

Peroxiredoxins, gerontogenes linking aging to genome instability and cancer

2012

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Age is the highest risk factor known for a large number of maladies, including cancers. However, it is unclear how aging mechanistically predisposes the organism to such diseases and which gene products are the primary targets of the aging process. Recent studies suggest that peroxiredoxins, antioxidant enzymes preventing tumor development, are targets of age-related deterioration and that bolstering their activity (e.g., by caloric restriction) extends cellular life span. This review focuses on how the peroxiredoxin functions (i.e., as peroxidases, signal transducers, and molecular chaperones) fit with contemporary theories of aging and whether peroxiredoxins could be targeted therapeutically in the treatment of age-associated cancers.

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Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype

Cited by 97 publications

References 104 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

Peroxiredoxins, gerontogenes linking aging to genome instability and cancer

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