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The Journal of Biological Chemistry May 1988Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn...
Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.
Topics: DNA Topoisomerases, Type I; Deoxyribonucleotides; Kinetics; Mutation; Phenotype; T-Phages; Temperature
PubMed: 2834366
DOI: No ID Found -
Chemical Research in Toxicology Apr 1999Both syn and anti dihydrodiol epoxides from 5-methylchrysene (5-MCDE) and 5,6-dimethylchrysene (5,6-DMCDE) were reacted under the same conditions with native DNA,...
Both syn and anti dihydrodiol epoxides from 5-methylchrysene (5-MCDE) and 5,6-dimethylchrysene (5,6-DMCDE) were reacted under the same conditions with native DNA, denatured DNA, and purine deoxyribonucleotides, and the products were quantified. The extents of reaction with the deoxyribonucleotides were consistently greater for 5,6-DMCDE than for 5-MCDE. The yield of adducts in the reaction with DNA ranged from being a few-fold to 50-fold greater than those found in the corresponding deoxyribonucleotide reactions for both 5-MCDE and 5,6-DMCDE. The DNA-dependent enhancement of product yield was greater for 5-MCDE than for 5,6-DMCDE with a few exceptions among cis and trans deoxyadenosine adducts. The most substantial differences in DNA-dependent enhancement were found for deoxyguanosine adducts; thus, steric hindrance between the 6-methyl group in the 5,6-DMCDE and the minor groove in the DNA double helix may account for the greater DNA-dependent enhancement found in the 5-MCDE reactions.
Topics: Carcinogens; Chrysenes; DNA; DNA Adducts; Deoxyribonucleotides; Structure-Activity Relationship
PubMed: 10207124
DOI: 10.1021/tx980228o -
The Journal of Biological Chemistry Nov 1962
Topics: Cytidine Diphosphate; Cytosine Nucleotides; Deoxycytosine Nucleotides; Deoxyribonucleotides; Escherichia coli
PubMed: 13973714
DOI: No ID Found -
Life Science Alliance Apr 2022Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication...
Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that negatively interacts with , which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.
Topics: Cell Cycle Proteins; Cells, Cultured; Chromosomal Proteins, Non-Histone; DEAD-box RNA Helicases; DNA Helicases; DNA Replication; Deoxyribonucleotides; Humans; Saccharomyces cerevisiae Proteins
PubMed: 35017203
DOI: 10.26508/lsa.202101153 -
Experimental Cell Research Apr 1989Alterations of the balanced supply of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, have dramatic genetic consequences for mammalian cells... (Review)
Review
Alterations of the balanced supply of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, have dramatic genetic consequences for mammalian cells including the induction of mutations, the sensitization to DNA damaging agents, and the production of gross chromosomal abnormalities. The use of recombinant DNA techniques has allowed the analysis of some of these effects and has revealed further mechanisms by which mammalian cells control the accuracy of DNA replication.
Topics: Alkylating Agents; Animals; Cell Line; DNA Replication; DNA, Recombinant; Deoxyribonucleotides; Mutation
PubMed: 2647496
DOI: 10.1016/0014-4827(89)90090-6 -
Applied Radiation and Isotopes :... Jul 2005Enzymatic synthesis of alpha-(32)P and alpha-(33)P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either gamma-(32)P adenosine...
Enzymatic synthesis of alpha-(32)P and alpha-(33)P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either gamma-(32)P adenosine triphosphate (gamma-ATP) or gamma-(32)P guanosine triphosphate (gamma-GTP). Subsequent removal of these ribonucleotides is essential for the preparation of chemically pure deoxyribonucleotides. Agarose-phenyl boronate columns, which bind specifically to cis-diol moieties, have been used for the removal of ribonucleotide contaminants. However, this involves column losses and additional radiation exposure. In the present work we describe a chemical method for the improvement of the chemical purity, based on the preferential oxidation of ribose sugars by periodate. The cis-diol moiety of ribose is specifically oxidised to the dialdehyde. The excess periodate ions were destroyed using ethylene glycol. The phosphate group was then cleaved by beta-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer gamma-(32)P ATP and alpha-(32)P dATP. Samples at various steps were analysed by TLC, autoradiography and HPLC. During the process ATP is oxidised whereas 2'-deoxyadenosine triphosphate (dATP) remains intact. The alpha-(32)P dATP synthesized by this process was assayed for its incorporation in lambda-DNA by the random priming method and was found to be effectively incorporated. The process developed is an efficient and convenient method for the preparation of chemically pure deoxyribonucleotides.
Topics: Autoradiography; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Deoxyribonucleotides; Enzymes; Oxidation-Reduction; Periodic Acid
PubMed: 15866449
DOI: 10.1016/j.apradiso.2005.03.002 -
Yi Chuan = Hereditas Feb 2022As an important precursor for DNA synthesis, the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are necessary raw materials for DNA replication,...
As an important precursor for DNA synthesis, the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are necessary raw materials for DNA replication, recombination, and repair in cells. The correct synthesis and integrity of DNA are important manifestations of the genome stability, so the stability of the dNTP library state is essential to maintain the stability of the genome and the cell. In terms of the quality of the dNTP library, the incorporation of some heterogeneous dNTPs, such as oxidized dNTPs, into DNA can easily cause base substitutions and even DNA breaks and rearrangements, which will greatly damage the stability of the genome. At the same time, the cell has also evolved the corresponding NTP pyrophosphatase to remove it, and to correct the damaged DNA and repair the DNA gap by forming a DNA damage repair network. In terms of the number of dNTP libraries, the imbalance of the dNTP concentration and ratio will also cause base and frameshift mutations, which will also cause genome instability. As a result, cells have evolved a huge enzyme-controlled network to carry them out under precise control. This article mainly reviews the potential harm of damage to dNTP library components in cells, the clearance of damaged dNTPs, the regulation on the balance between dNTP library components, and finally discusses clinical diseases related to dNTP library homeostasis. It provides insights on the research of the correlation between the stability of the cellular dNTP library and the genome, and finally provides some theoretical basis for the treatment of related diseases.
Topics: DNA Replication; Deoxyribonucleotides; Genome; Genomic Instability; Homeostasis; Humans
PubMed: 35210212
DOI: 10.16288/j.yczz.21-211 -
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 -
Advances in Space Research : the... 1986Unlike ribose chemistry, the chemistry of 2-deoxyribose precludes its formation or at least its incorporation into nucleotides under accepted "primordial soup"...
Unlike ribose chemistry, the chemistry of 2-deoxyribose precludes its formation or at least its incorporation into nucleotides under accepted "primordial soup" conditions; therefore RNA and DNA could not develop in parallel during the evolution of protocells. However, deoxyribonucleotides might have been formed abiotically by direct reduction of ribonucleotides in a primitive version of the biochemical pathway. This sequence of events, in which DNA lagged behind RNA in the assembly of genetic information for an unknown--probably short--period of time is suggested by the primitive traits (i.e., nucleotide binding, thiol redox chemistry, and metal ion catalysis) of present-day enzyme systems of deoxyribonucleotide biosynthesis. The reaction should be amenable to experimental study.
Topics: Carbohydrates; DNA; Deoxyribonucleotides; Deoxyribose; Evolution, Molecular; Hydroxyl Radical; Origin of Life; Ribonucleotide Reductases
PubMed: 11537241
DOI: 10.1016/0273-1177(86)90272-3 -
Methods in Molecular Biology (Clifton,... 2019Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability....
Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and altered regulation of RNR leads to imbalanced dNTP pools. Increased dNTP levels are mutagenic and have the potential to interfere with pathways that are involved in DNA replication, repair and DNA damage control. However, the mechanisms through which altered dNTP pools affect these pathways are poorly understood. Nonetheless, altered dNTP pools have been identified in a number of cellular contexts, including cancer. In order to interpret and analyze the effects of altered dNTP pools, we need quantitative information about dNTP pools in different genetic and environmental contexts in vivo. Here we describe a high-throughput fluorescence-based assay that uses a qPCR-based approach to quantify dNTP levels for use with Saccharomyces cerevisiae extracts.
Topics: DNA Repair; Deoxyribonucleotides; Fluorescence; High-Throughput Screening Assays; Mutagenesis; Real-Time Polymerase Chain Reaction; Ribonucleotide Reductases; Saccharomyces cerevisiae
PubMed: 31127572
DOI: 10.1007/978-1-4939-9500-4_6