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Scientific Reports Nov 2015Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular...
Endogenous ribonucleotides and deoxyribonucleotides are essential metabolites that play important roles in a broad range of key cellular functions. Their intracellular levels could also reflect the action of nucleoside analogues. We investigated the effects of 5-fluorouracil (5-FU) on ribonucleotide and deoxyribonucleotide pool sizes in cells upon exposure to 5-FU for different durations. Unsupervised and supervised artificial neural networks were compared for comprehensive analysis of global responses to 5-FU. As expected, deoxyuridine monophosphate (dUMP) increased after 5-FU incubation due to the inhibition of thymine monophosphate (TMP) synthesis. Interestingly, the accumulation of dUMP could not lead to increased levels of deoxyuridine triphosphate (dUTP) and deoxyuridine diphosphate (dUDP). After the initial fall in intracellular deoxythymidine triphosphate (TTP) concentration, its level recovered and increased from 48 h exposure to 5-FU, although deoxythymidine diphosphate (TDP) and TMP continued to decrease compared with the control group. These findings suggest 5-FU treatment caused unexpected changes in intracellular purine polls, such as increases in deoxyadenosine triphosphate (dATP), adenosine-triphosphate (ATP), guanosine triphosphate (GTP) pools. Further elucidation of the mechanism of action of 5-FU in causing these changes should enhance development of strategies that will increase the anticancer activity of 5-FU while decreasing its resistance.
Topics: Adenine Nucleotides; Cell Survival; Chromatography, Liquid; Cytosine Nucleotides; Deoxyribonucleotides; Fluorouracil; Guanine Nucleotides; Hep G2 Cells; Humans; Inhibitory Concentration 50; Metabolic Networks and Pathways; Models, Biological; Neural Networks, Computer; Ribonucleotides; Tandem Mass Spectrometry; Thymine Nucleotides; Uracil Nucleotides
PubMed: 26578061
DOI: 10.1038/srep16861 -
Nucleosides, Nucleotides & Nucleic Acids May 2011The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double... (Review)
Review
The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10(-7)) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm.
Topics: Animals; Biological Transport; Cell Transformation, Neoplastic; Cytoplasm; DNA, Mitochondrial; Deoxyribonucleotides; Humans; Mitochondria
PubMed: 21774628
DOI: 10.1080/15257770.2011.586955 -
Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-D-glucose epimerization in Pseudomonas putida KT2440.Scientific Reports Jun 2021L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite...
L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-β-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.
Topics: Carbohydrate Epimerases; Catalysis; Databases, Factual; Deoxyglucose; Deoxyribonucleotides; Escherichia coli; Gene Library; Nucleoside Diphosphate Sugars; Protein Conformation; Pseudomonas putida; Structure-Activity Relationship; Thymine Nucleotides
PubMed: 34099824
DOI: 10.1038/s41598-021-91421-x -
Methods in Molecular Biology (Clifton,... 2017DNA damage response has been characterized as an important mediator of senescence phenotypes induced by activated oncogenes in normal human cells. Depletion of...
DNA damage response has been characterized as an important mediator of senescence phenotypes induced by activated oncogenes in normal human cells. Depletion of intracellular deoxyribonucleotide pools has been recently recognized as one of the major causes for DNA damage in these cells. Cells undergoing oncogene-induced senescence display decreased expression of several rate-limiting enzymes involved in the biosynthesis of deoxyribonucleotides, including thymidylate synthase (TS) and ribonucleotide reductase (RR). Individual depletion of these enzymes leads to premature senescence. Reciprocally, ectopic expression of TS and RR or addition of deoxyribonucleosides resulted in suppression of senescence phenotypes in normal or tumor cells caused by overexpression of activated HRAS or depletion of C-MYC, respectively. Therefore, in the current chapter, we will describe a methodology for the quantitative measurement of nucleotide pools in senescent cells.
Topics: Cell Line; Cellular Senescence; Chromatography, High Pressure Liquid; DNA Damage; Deoxyribonucleotides; Fibroblasts; Gene Expression; Genes, myc; Humans; Nucleotides; Oncogenes; Ribonucleotide Reductases; Thymidylate Synthase
PubMed: 27812878
DOI: 10.1007/978-1-4939-6670-7_16 -
The Journal of Biological Chemistry Mar 2013A large number of nucleoside analogues and 2'-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the...
A large number of nucleoside analogues and 2'-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5'-monophosphate-2'-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells.
Topics: Animals; Bacterial Proteins; Deoxyribonucleotides; Lactobacillus; Pentosyltransferases; Rats; Recombinant Fusion Proteins
PubMed: 23325804
DOI: 10.1074/jbc.M112.446492 -
The Journal of Biological Chemistry Nov 2019Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells...
Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells and with purified proteins. In this study, Tran use technology to inactivate RNR large subunit expression in heart and skeletal muscle of mouse embryos. Analysis of these mutants paints a picture of dNTP regulation in whole animals quite different from that seen in studies of purified proteins and cultured cells.
Topics: Animals; DNA Replication; Deoxyribonucleotides; Heart; Mice; Ribonucleotide Reductases
PubMed: 31676554
DOI: 10.1074/jbc.H119.011335 -
Nucleic Acids Research Feb 2022Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for...
Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.
Topics: Adenosine Diphosphate; Chromatography, High Pressure Liquid; DNA; Deoxyribonucleotides; Reproducibility of Results; Ribonucleotides
PubMed: 34850106
DOI: 10.1093/nar/gkab1117 -
The Journal of Biological Chemistry Aug 2001Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3'-hydroxyl ends of DNA strands in a template-independent manner and...
Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3'-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.
Topics: Catalysis; DNA Nucleotidylexotransferase; Deoxyribonucleotides; Recombination, Genetic; Ribonucleotides
PubMed: 11406636
DOI: 10.1074/jbc.M105272200 -
Nucleic Acids Research Jan 2022Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to...
Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cellular levels of the four canonical building blocks of DNA and of exotic dNTPs, as well. In addition to the measured quantity, dNTPpoolDB contains ample information on sample source, dNTP quantitation methods and experimental conditions including any treatments and genetic manipulations. Functions such as the advanced search offering multiple choices from custom-built controlled vocabularies in 15 categories in parallel, the pairwise comparison of any chosen pools, and control-treatment correlations provide users with the possibility to quickly recognize and graphically analyse changes in the dNTP pools in function of a chosen parameter. Unbalanced dNTP pools, as well as the balanced accumulation or depletion of all four dNTPs result in genomic instability. Accordingly, key roles of dNTP pool homeostasis have been demonstrated in cancer progression, development, ageing and viral infections among others. dNTPpoolDB is designated to promote research in these fields and fills a longstanding gap in genome metabolism research.
Topics: DNA Replication; Data Curation; Databases, Genetic; Deoxyribonucleotides; Genomic Instability; Humans; Neoplasms
PubMed: 34643700
DOI: 10.1093/nar/gkab910 -
Nucleic Acids Research Nov 2023The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases...
The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.
Topics: Catalytic Domain; Crystallography, X-Ray; Deoxyribonucleotides; DNA; DNA Polymerase II; Ribonucleotides; Saccharomyces cerevisiae
PubMed: 37819038
DOI: 10.1093/nar/gkad817