Quantitation of cellular deoxynucleoside triphosphates

Ferraro, Paola; Franzolin, Elisa; Pontarin, Giovanna; Reichard, Peter; Bianchi, Vera

doi:10.1093/nar/gkp1141

Cited by 147 publications

(204 citation statements)

References 31 publications

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“…The repression of E2F1 leads to down-regulation of RNR2 gene expression, which blocks de novo dNTP biosynthesis, thus impairing reverse transcription. In addition to impairing HIV cDNA synthesis, dNTP depletion following RNR2 down-regulation may also favor misincorporation of cellular rNMPs that are efficiently incorporated into HIV-1 DNA in macrophages (65,66).…”

Section: Discussionmentioning

confidence: 99%

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

Allouch

David

Amie

et al. 2013

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

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Macrophages are a major target cell for HIV-1, and their infection contributes to HIV pathogenesis. We have previously shown that the cyclin-dependent kinase inhibitor p21 inhibits the replication of HIV-1 and other primate lentiviruses in human monocyte-derived macrophages by impairing reverse transcription of the viral genome. In the attempt to understand the p21-mediated restriction mechanisms, we found that p21 impairs HIV-1 and simian immunodeficiency virus (SIV)mac reverse transcription in macrophages by reducing the intracellular deoxyribonucleotide (dNTP) pool to levels below those required for viral cDNA synthesis by a SAM domain and HD domain-containing protein 1 (SAMHD1)-independent pathway. We found that p21 blocks dNTP biosynthesis by down-regulating the expression of the RNR2 subunit of ribonucleotide reductase, an enzyme essential for the reduction of ribonucleotides to dNTP. p21 inhibits RNR2 transcription by repressing E2F1 transcription factor, its transcriptional activator. Our findings unravel a cellular pathway that restricts HIV-1 and other primate lentiviruses by affecting dNTP synthesis, thereby pointing to new potential cellular targets for anti-HIV therapeutic strategies.

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Section: Discussionmentioning

confidence: 99%

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

Allouch

David

Amie

et al. 2013

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

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“…In particular, it is likely that the hypermutators accumulate mutated (misfolded) proteins that can trigger defined stress responses, contributing to the poor growth and mutability (37,39,40,42,44). In contrast to the high concentrations of the cellular rNTPs, which are generally in the millimolar range, those of the dNTPs are in the low-to mid-micromolar range (45)(46)(47)(48)(49). Thus, although high levels of the dNTPs would presumably facilitate rapid DNA replication, it is clear that such use of high dNTP levels would be a bad strategy from a fidelity perspective.…”

Section: Discussionmentioning

confidence: 99%

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

Ahluwalia

Schaaper

2013

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

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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,...

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“…After drying, radioactivity on the papers was measured in a liquid scintillation counter (Beckman). In case of dCTP measurement we used Taq polymerase, (RedTaq, Sigma), as Klenow polymerase is capable of incorporating CTP and GTP from nucleotide extracts [41]. Incubation was carried out at 48°C for 1 hour.…”

Section: Determination Of the Pyrimidine Pool Sizementioning

confidence: 99%

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

et al. 2015

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Quantitation of cellular deoxynucleoside triphosphates

Cited by 147 publications

References 31 publications

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

p21-mediated RNR2 repression restricts HIV-1 replication in macrophages by inhibiting dNTP biosynthesis pathway

Hypermutability and error catastrophe due to defects in ribonucleotide reductase

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

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