-
The Journal of Biological Chemistry Dec 1982A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at...
A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
Topics: Centrifugation, Density Gradient; Cytosol; DNA Replication; Deoxyribonucleotides; Escherichia coli; Kinetics; Multienzyme Complexes; T-Phages; Virus Replication
PubMed: 6757252
DOI: No ID Found -
Molecular Cancer Sep 2015Regulation of intracellular deoxynucleoside triphosphate (dNTP) pool is critical to genomic stability and cancer development. Imbalanced dNTP pools can lead to enhanced... (Review)
Review
Regulation of intracellular deoxynucleoside triphosphate (dNTP) pool is critical to genomic stability and cancer development. Imbalanced dNTP pools can lead to enhanced mutagenesis and cell proliferation resulting in cancer development. Therapeutic agents that target dNTP synthesis and metabolism are commonly used in treatment of several types of cancer. Despite several studies, the molecular mechanisms that regulate the intracellular dNTP levels and maintain their homeostasis are not completely understood. The discovery of SAMHD1 as the first mammalian dNTP triphosphohydrolase provided new insight into the mechanisms of dNTP regulation. SAMHD1 maintains the homeostatic dNTP levels that regulate DNA replication and damage repair. Recent progress indicates that gene mutations and epigenetic mechanisms lead to downregulation of SAMHD1 activity or expression in multiple cancers. Impaired SAMHD1 function can cause increased dNTP pool resulting in genomic instability and cell-cycle progression, thereby facilitating cancer cell proliferation. This review summarizes the latest advances in understanding the importance of dNTP metabolism in cancer development and the novel function of SAMHD1 in regulating this process.
Topics: Cell Proliferation; DNA Replication; Deoxyribonucleotides; Genomic Instability; Humans; Monomeric GTP-Binding Proteins; Mutation; Neoplasms; SAM Domain and HD Domain-Containing Protein 1
PubMed: 26416562
DOI: 10.1186/s12943-015-0446-6 -
Journal of Molecular Evolution Aug 1998Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one...
Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one accepts the explanatory strength of the RNA world model, it is clear that DNA molecules arose in the RNA world at some stage during the early evolution of cells. I suggest that competition for limited and valuable resources such as nucleotides, amino acids, and sugars made an early appearance among RNA cells, RNA viruses, viroids, and RNA plasmids. Structural and functional similarities between the different types of polymerases favor the simple hypothesis that the first RTs were RNA polymerase mutants that preferentially joined together preexisting deoxyribonucleotide triphosphates (dNTPs) using RNA templates. What was the role of dNTPs inside cells before DNA was synthesized and tested by natural selection? The oxygen atom that is removed by the reductase is of crucial importance to many ribozyme functions, since the 2'-OH is a strong nucleophile that forms transitional states during catalysis. Consequently, a RNR may have been used by cellular parasites to inhibit ribozyme action. Thus, DNA may have been, initially, an inert by-product of retrotranscription in lineages that acquired RTs and could synthesize DNA molecules using cellular RNA templates to detoxify the intracellular environment. DNA was useless as template until a transcriptase (DNA-dependent RNA polymerase) evolved that could copy (-)DNA to reconstitute the (+)RNA genome, indeed a successful way of confronting ribonuclease threats in the RNA world.
Topics: DNA; DNA-Directed RNA Polymerases; Deoxyribonucleotides; Evolution, Molecular; Models, Theoretical; Mutation; RNA, Catalytic; RNA-Directed DNA Polymerase; Ribonucleotide Reductases
PubMed: 9694660
DOI: 10.1007/pl00006368 -
Pharmacology & Therapeutics 1990Inhibitory and substrate properties of analogs of deoxyribonucleoside triphosphates toward DNA polymerases are reviewed. A general introduction is followed by a... (Review)
Review
Inhibitory and substrate properties of analogs of deoxyribonucleoside triphosphates toward DNA polymerases are reviewed. A general introduction is followed by a description of DNA polymerases and the reaction that they catalyze, and sites at which substrate analogs may inhibit them. Effects of modifications in the major family of compounds, nucleotide derivatives, at the base, sugar and triphosphate portions of the molecule, are summarized with respect to retention of substrate properties and generation of inhibitory properties. Structure-activity relationships and the basis of selectivity in the second family of compounds, deoxyribonucleotide mimics, are also presented. Conclusions are drawn regarding the structural basis of inhibitor selectivity and mechanism, relationship between in vitro and in vivo effects of inhibitors, and the promise of inhibitors as probes for study of active sites of DNA polymerases.
Topics: Animals; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Humans; Nucleic Acid Synthesis Inhibitors; Substrate Specificity
PubMed: 2290857
DOI: 10.1016/0163-7258(90)90066-b -
Scientific Reports Jan 2020The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and...
The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 10 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.
Topics: Click Chemistry; Copper; Cycloaddition Reaction; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; Rhodamines; Staining and Labeling; Thymine Nucleotides
PubMed: 31953472
DOI: 10.1038/s41598-020-57463-3 -
Bioorganic & Medicinal Chemistry Aug 2003Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and...
Synthesis and properties of oligo-2'-deoxyribonucleotides containing internucleotidic phosphoramidate linkages modified with pendant groups ending with either two amino or two hydroxyl functions.
Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2'-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2'-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences. Incorporating the modifications into the third oligonucleotide strands has little effect on the structure of the triplexes. At pH 7, the incorporation of two, three or four modified phosphate groups into the third strands stabilizes the triplexes, as compared to the unmodified oligonucleotide. Stronger stabilization was observed with compounds containing linkers ending with amino functions. Stability increases with the number of modifications without being fully additive. This might be due to the different environments of the phosphate groups inside the sequence.
Topics: Amides; Base Sequence; Circular Dichroism; Deoxyribonucleotides; Hydroxylation; Isomerism; Molecular Structure; Nucleic Acid Denaturation; Phosphoric Acids; Spectrophotometry; Transition Temperature; Ultraviolet Rays
PubMed: 12878143
DOI: 10.1016/s0968-0896(03)00273-6 -
Nucleic Acids Research Sep 2019A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read...
A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
Topics: DNA-Directed DNA Polymerase; Deoxyribonucleosides; Deoxyribonucleotides; High-Throughput Nucleotide Sequencing; Limit of Detection; Microscopy, Fluorescence; Oligodeoxyribonucleotides; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 31318971
DOI: 10.1093/nar/gkz611 -
Progress in Nucleic Acid Research and... 2002For growth under oxygen-free atmosphere, some strict or facultative anaerobes depend on a class III ribonucleotide reductase for the synthesis of deoxyribonucleotides,... (Review)
Review
For growth under oxygen-free atmosphere, some strict or facultative anaerobes depend on a class III ribonucleotide reductase for the synthesis of deoxyribonucleotides, the DNA precursors. Prototypes for this class of enzymes are ribonucleotide reductases from Escherichia coli and bacteriophage T4. This review article describes their structural and mechanistic properties as well as their complex allosteric regulation. Their evolutionnary relationship to class I and class II ribonucleotide reductases is also discussed.
Topics: Amino Acid Sequence; Anaerobiosis; Bacteria, Anaerobic; Bacterial Proteins; DNA; Deoxyribonucleotides; Escherichia coli Proteins; Gene Expression Regulation; Molecular Sequence Data; RNA; Ribonucleotide Reductases; Viral Proteins
PubMed: 12206460
DOI: 10.1016/s0079-6603(02)72068-0 -
Journal of the American Chemical Society Apr 2003We report here the synthesis and binding studies of oligo-2'-deoxyribonucleotides (ODNs) containing 2'-deoxyuridines, modified at the 5-position by linkers ending with...
We report here the synthesis and binding studies of oligo-2'-deoxyribonucleotides (ODNs) containing 2'-deoxyuridines, modified at the 5-position by linkers ending with either one or two guanidinium groups. Comparison was made with ODNs containing 2'-deoxyuridines modified at the 5-position with linkers ending with either two or one amino groups. One or two modified 2'-deoxyuridines were incorporated into pyrimidine strands, and their influence on the stability of duplex (with both DNA and RNA targets) and triplex structures was studied. The strongest stabilization was obtained with modified ODNs containing guanidinium groups. This result confirms that the reduction of the global negative charge number on one strand is an important parameter in the stability of duplex and triplex structures.
Topics: Cross-Linking Reagents; DNA; Deoxyribonucleotides; Deoxyuridine; Guanidine; Oligonucleotides; Thermodynamics
PubMed: 12683800
DOI: 10.1021/ja029467v -
The Journal of Biological Chemistry May 1977The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state...
Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.
The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.
Topics: Coliphages; DNA Replication; DNA, Viral; Deoxycytidine Monophosphate; Deoxyribonucleotides; Escherichia coli; Kinetics; Mutation; Thymine Nucleotides; Uracil
PubMed: 323259
DOI: No ID Found