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RNA (New York, N.Y.) Aug 1997The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant...
The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.
Topics: Base Sequence; DNA-Directed RNA Polymerases; Deoxyribonucleotides; Models, Molecular; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; RNA, Transfer; RNA, Transfer, Asp; RNA, Transfer, Met; Structure-Activity Relationship; Transcription, Genetic; Viral Proteins
PubMed: 9257648
DOI: No ID Found -
Journal of Biochemistry Jul 1976In order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16...
In order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinucleoside phosphates (XpY's) as substrates at pH 5.5 and 25 degrees. The maximum rates of transesterification of GpY's were in the range of 10,000 to 2,800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY. The Ki values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhibitory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the Ki values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribonucleosides was in the order dT, dA, dG, and dC.
Topics: Aspergillus; Deoxyribonucleosides; Deoxyribonucleotides; Endonucleases; Kinetics; Ribonucleases; Ribonucleosides; Ribonucleotides; Structure-Activity Relationship
PubMed: 965365
DOI: 10.1093/oxfordjournals.jbchem.a131255 -
European Journal of Biochemistry Sep 1980Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and...
Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and maximum deoxyribonucleoside 5'-triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2-3 pmol/10(6) cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP leads to dNDP leads to dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 micrograms 5-fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5-Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.
Topics: Cell Cycle; Chlorophyta; DNA; Deoxyribonucleotides; Floxuridine; Light; Periodicity; Ribonucleoside Diphosphate Reductase; Thymine Nucleotides
PubMed: 6449366
DOI: 10.1111/j.1432-1033.1980.tb04843.x -
Journal of Virology Oct 2016Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by...
UNLABELLED
Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner.
IMPORTANCE
Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.
Topics: Cells, Cultured; Cytomegalovirus; DNA, Viral; Deoxyribonucleotides; Humans; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Retinoblastoma Protein; Viral Plaque Assay; Virus Replication
PubMed: 27440891
DOI: 10.1128/JVI.00731-16 -
Nucleosides, Nucleotides & Nucleic Acids 2023We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl...
We describe a new demethylation method for dimethyl phosphonate esters using sodium ethanethiolate. The new procedure allows demethylation of nucleoside dimethyl phosphonate esters without 1'-α-anomerization, providing an improved synthesis of 5'-methylene substituted 2',5'-deoxynucleotides.
Topics: Esters; Organophosphonates; Deoxyribonucleotides; Demethylation
PubMed: 36629008
DOI: 10.1080/15257770.2023.2166064 -
Proceedings of the National Academy of... Apr 1980A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells...
A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [(3)H]dUrd contained dUMP in DNA in readily detectable amounts ( approximately 0.8 pmol of dUMP per mumol of total DNA nucleotide), and this was increased approximately 3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/mumol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.
Topics: Cell Line; DNA; DNA Replication; Deoxyribonucleotides; Deoxyuracil Nucleotides; Humans; Methotrexate
PubMed: 6929529
DOI: 10.1073/pnas.77.4.1956 -
The EMBO Journal Aug 2020Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their...
Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.
Topics: Cell Line; DNA Repair; Deoxyribonucleotides; Humans; Immunoglobulin Class Switching; SAM Domain and HD Domain-Containing Protein 1
PubMed: 32511795
DOI: 10.15252/embj.2019102931 -
Nucleic Acids Research Mar 2002Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein-DNA and 14 drug-DNA complexes. It was found...
Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein-DNA and 14 drug-DNA complexes. It was found that only purine N3, pyrimidine O2, guanine N2 and deoxyribose O4' are involved in the interactions, and that contacts to N3 and O2 are most frequent and more polar than contacts to O4'. Many protein contacts are mediated by water, possibly to increase the DNA effective surface. Fewer water-mediated contacts are observed in drug complexes. The distributions of ligands around N3 are significantly more compact than around O2, and distributions of water molecules are the most compact. Distributions around O4' are more diffuse than for the base atoms but most distributions still have just one binding site. Ligands bind to N3 and O2 atoms in analogous positions, and simultaneous binding to N3 and N2 in guanines is extremely rare. Contacts with two consecutive nucleotides are much more frequent than base-sugar contacts within one nucleotide. The probable reason for this is the large energy of deformation of hydrogen bonds for the one nucleotide motif. Contacts of Arg, the most frequent amino acid ligand, are stereochemically indistinguishable from the binding of the remaining amino acids except asparagine (Asn) and phenylalanine (Phe). Asn and Phe bind in distinct ways, mostly to a deformed DNA, as in the complexes of TATA-box binding proteins. DNA deformation concentrates on dinucleotide regions with a distinct deformation of the delta and epsilon backbone torsion angles for the Asn and delta, epsilon, zeta and chi for the Phe-contacted regions.
Topics: Amino Acids; Binding Sites; Crystallography, X-Ray; DNA; DNA-Binding Proteins; Deoxyribonucleotides; Drug Design; Hydrogen Bonding; Ligands; Macromolecular Substances; Models, Chemical; Nitrogen; Nucleic Acid Conformation; Oxygen; Protein Binding; Water
PubMed: 11861910
DOI: 10.1093/nar/30.5.1182 -
BMC Medical Research Methodology Nov 2019Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed... (Comparative Study)
Comparative Study
A comparison of machine learning techniques for classification of HIV patients with antiretroviral therapy-induced mitochondrial toxicity from those without mitochondrial toxicity.
BACKGROUND
Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed drug-associated toxicity. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of ART regimens. NRTIs compete with endogenous deoxyribonucleotide triphosphates (dNTPs) in incorporation into elongating DNA chain resulting in their cytotoxic or antiviral effect. Thus, the efficacy of NRTIs could be affected by direct competition with endogenous dNTPs and/or feedback inhibition of their metabolic enzymes. In this paper, we assessed whether the levels of ribonucleotides (RN) and dNTP pool sizes can be used as biomarkers in distinguishing between HIV-infected patients with ART-induced mitochondrial toxicity and HIV-infected patients without toxicity.
METHODS
We used data collected through a case-control study from 50 subjects. Cases were defined as HIV-infected individuals with clinical and/or laboratory evidence of mitochondrial toxicity. Each case was age, gender, and race matched with an HIV-positive without evidence of toxicity. We used a range of machine learning procedures to distinguish between patients with and without toxicity. Using resampling methods like Monte Carlo k-fold cross validation, we compared the accuracy of several machine learning algorithms applied to our data. We used the algorithm with highest classification accuracy rate in evaluating the diagnostic performance of 12 RN and 14 dNTP pool sizes as biomarkers of mitochondrial toxicity.
RESULTS
We used eight classification algorithms to assess the diagnostic performance of RN and dNTP pool sizes distinguishing HIV patients with and without NRTI-associated mitochondrial toxicity. The algorithms resulted in cross-validated classification rates of 0.65-0.76 for dNTP and 0.72-0.83 for RN, following reduction of the dimensionality of the input data. The reduction of input variables improved the classification performance of the algorithms, with the most pronounced improvement for RN. Complex tree-based methods worked the best for both the deoxyribose dataset (Random Forest) and the ribose dataset (Classification Tree and AdaBoost), but it is worth noting that simple methods such as Linear Discriminant Analysis and Logistic Regression were very competitive in terms of classification performance.
CONCLUSIONS
Our finding of changes in RN and dNTP pools in participants with mitochondrial toxicity validates the importance of dNTP pools in mitochondrial function. Hence, levels of RN and dNTP pools can be used as biomarkers of ART-induced mitochondrial toxicity.
Topics: Algorithms; Anti-Retroviral Agents; Biomarkers; Case-Control Studies; Deoxyribonucleotides; Dideoxynucleotides; HIV Infections; Humans; Machine Learning; Ribonucleotides
PubMed: 31775643
DOI: 10.1186/s12874-019-0848-z -
Journal of Molecular Evolution Dec 2014All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve...
All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route--the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production.
Topics: Aldehyde-Lyases; Biological Evolution; DNA; DNA Replication; Deoxyribonucleotides; Eukaryota; Origin of Life; Prokaryotic Cells; Ribonucleotide Reductases; Time Factors
PubMed: 25425102
DOI: 10.1007/s00239-014-9656-6