Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Sánchez, Arancha; Sharma, Sushma; Rozenzhak, Sophie; Roguev, Assen; Krogan, Nevan J.; Chabes, Andrei; Russell, Paul

doi:10.1128/mcb.01062-12

Cited by 40 publications

(47 citation statements)

References 70 publications

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“…The effects on cell fitness are similar to what we observed. Consistent with our model, dcd1Δ cells expressing hsv-tk + increase dTTP pools, relieving growth defects and dcd1Δ sensitivities to UV, HU, and bleomycin (Sanchez et al 2012).…”

Section: Discussionsupporting

confidence: 66%

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

et al. 2013

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Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-29-deoxyuridine (BrdU) and 5-ethynyl-29-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

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Section: Discussionsupporting

confidence: 66%

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

et al. 2013

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“…In addition, not all dNTP imbalances correlate with increased mutation rates. For example, inactivation of dCMP deaminase (dcd1Δ) in S. cerevisiae resulted in 3-fold reduction in dTTP pool and about 30-fold increased in dCTP levels, without any consequences on mutation rates at the URA4 reporter (92).…”

Section: Discussionmentioning

confidence: 99%

Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis

Schmidt

Reyes

Gries

et al. 2017

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

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Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in Saccharomyces cerevisiae using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (GLN3, URA7, and SHM2) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.DNA replication fidelity | mismatch repair | CTP biosynthesis | DNA polymerases | dNTP pool imbalance T he fidelity of DNA replication is strongly influenced by three processes (1-3): (i) nucleotide selectivity, wherein replicative DNA polymerases select the correct dNTP to be incorporated; (ii) DNA polymerase proofreading activity, which excises wrongly incorporated nucleotides by using the DNA polymerase 3′-to-5′ exonuclease activity; and (iii) mismatch repair (MMR) (4, 5), a DNA replication-coupled repair mechanism (6, 7), which corrects errors that escaped proofreading. Furthermore, the balance and overall concentration of dNTPs not only affect nucleotide selection but also influence DNA polymerase proofreading activity (8). A central player in the biosynthesis of dNTPs is the ribonucleotide reductase (RNR) holoenzyme, which catalyzes the reduction of NDPs to dNDPs (9, 10). In Saccharomyces cerevisiae, RNR is composed of two identical Rnr1 large subunits that associate with two smaller subunits represented by Rnr2 and Rnr4 (11,12). In addition, a second large subunit has been identified (Rnr3), which is induced upon DNA damage and when overexpressed can rescue the rnr1 lethal phenotype (13).The interplay between nucleotide selectivity, DNA polymerase proofreading activity, and MM...

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“…17,18), deaminating dCTP to dUTP, which is then hydrolyzed to dUMP. In most Gram positive bacteria, and in yeast and higher cells, this step occurs at the monophosphate level, deaminating dCMP to dUMP (19,20; see 2). The direct effect of deleting the dcd gene is the buildup of dCTP pools and, in some cases, the lowering of dTTP pools (2–4,18,21–22).…”

Section: Introductionmentioning

confidence: 99%

Extreme dNTP pool changes and hypermutability in dcd ndk strains

Tse¹,

Kang²,

Yuan³

et al. 2016

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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SUMMARY Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of E. coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.

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Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Cited by 40 publications

References 70 publications

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis

Extreme dNTP pool changes and hypermutability in dcd ndk strains

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