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Developmental Cell Aug 2017Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about...
Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. We examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized "on the go." The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis.
Topics: Animals; Deoxyribonucleotides; Drosophila; Embryonic Development; Feedback, Physiological
PubMed: 28735680
DOI: 10.1016/j.devcel.2017.06.013 -
Analytical Chemistry Nov 2011The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned...
The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.
Topics: Base Sequence; Candida; DNA, Fungal; Deoxyribonucleotides; Electrodes; Enzymes; Microfluidic Analytical Techniques; Sequence Analysis, DNA; Templates, Genetic
PubMed: 21932784
DOI: 10.1021/ac201416j -
Cell Proliferation Jul 1993Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle...
Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphosphates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (> 90,000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wave-lengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was > or = 99% and replicate extractions within or between samples were highly reproducible (SD < 5%). The single step method described minimizes potential losses associated with post-extraction manipulation and provides the capability to examine alterations in nucleotide precursor-product metabolism under various physiological and pharmacological conditions.
Topics: Adenosine Diphosphate; Animals; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; Deoxyribonucleotides; Freezing; Liver; Male; Rats; Rats, Inbred F344; Ribonucleotides; Spleen; Tissue Extracts
PubMed: 8343561
DOI: 10.1111/j.1365-2184.1993.tb00328.x -
The Journal of Biological Chemistry Oct 1963
Topics: Deoxyribonucleotides; Deoxyuracil Nucleotides; Escherichia coli; Phosphoric Monoester Hydrolases; Phosphotransferases; Research; Uracil Nucleotides
PubMed: 14085395
DOI: No ID Found -
Basic Life Sciences 1985Ribonucleotide reductase plays a crucial role in the maintenance of dNTP pools. A molecular model for the allosteric regulation of the enzyme is substantiated by the... (Review)
Review
Ribonucleotide reductase plays a crucial role in the maintenance of dNTP pools. A molecular model for the allosteric regulation of the enzyme is substantiated by the effects of nucleosides or inhibitors on pool sizes in intact cells as well as by manifestations of mutations of the reductase and other enzymes involved in deoxynucleoside metabolism. Studies of the dynamics of dNTP pools in 3T6 cells suggest compartmentation of dCTP pools, with perferential DNA synthesis from a pool labeled from cytidine. In contrast, no kinetic evidence for more than one dTTP pool was obtained. Our experiments also suggest that all dTTP formed in growing 3T6 cells is used up for DNA synthesis.
Topics: Allosteric Regulation; Animals; Binding Sites; Cell Cycle; Cell Line; DNA; Deoxyadenosines; Deoxycytosine Nucleotides; Deoxyribonucleotides; Kinetics; Macromolecular Substances; Models, Biological; Mutation; Protein Conformation; Ribonucleotide Reductases; Substrate Specificity; Thymidine; Thymine Nucleotides
PubMed: 3888178
DOI: 10.1007/978-1-4613-2449-2_3 -
Journal of Biomolecular Structure &... Feb 2005We have analyzed the relative stabilities and Gibbs tautomeric free energy for tautomeric transitions of neutral 2'-deoxyribonucleotides and its mono- and di-protonated... (Comparative Study)
Comparative Study
We have analyzed the relative stabilities and Gibbs tautomeric free energy for tautomeric transitions of neutral 2'-deoxyribonucleotides and its mono- and di-protonated forms. Geometry optimizations of these nucleic acid constituents have been performed at the DFT/B3LYP level using the standard 6-31G(d) basis set. The prediction of relative stabilities, Gibbs tautomeric free energy has been made at the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level of theory. For each nucleoside four major conformers, i.e., north/anti, north/syn, south/anti, and south/syn have been taken into consideration. We have found the substantial effect of the uncompensated charge on the relative stability of 2'-deoxyribonucleotides. In particular, when the charge of 2'-deoxyribonucleotide anions is completely compensated by protons, the syn conformations have been found to be the global minima due to stabilization provided by intramolecular hydrogen bonds. However, the negative charge that appears due to the successive removal of the protons from the phosphate group destabilizes these syn conformations and stabilizes preferably the south/anti conformations (except of 2'-deoxyguanosine phosphate). Only 2'-deoxyribonucleotides, possessing south/anti and north/anti orientations, containing guanine and cytosine can contribute significantly to the rate of spontaneous point mutations due to the formation of biologically relevant amounts of 'rare' tautomers. However, we found strong influence of uncompensated negative charge for 2'-deoxyribonucleotides which possess syn conformations. Finally we have found that the proton transfer could result in the spontaneous change of 2'-deoxyribonucleotides conformations. We conclude that this phenomenon could be considered as a new way for the stabilization of 'rare' isomers for such DNA bases as cytosine and thymine.
Topics: Anions; Computer Simulation; Cytosine; Deoxyribonucleotides; Drug Stability; Guanine; Hydrogen Bonding; Molecular Conformation; Molecular Structure; Phosphates; Point Mutation; Protons; Quantum Theory; Thermodynamics
PubMed: 15588107
DOI: 10.1080/07391102.2005.10507015 -
Current Drug Targets. Infectious... May 2001In the past years, a variety of 4'-C-substituted-2'-deoxynucleosides (4'SdNs) were designed, synthesized, and examined as potential therapeutics against human... (Review)
Review
In the past years, a variety of 4'-C-substituted-2'-deoxynucleosides (4'SdNs) were designed, synthesized, and examined as potential therapeutics against human immunodeficiency virus (HIV) infection and certain such analogues proved to exert potent activity against HIV-1 in vitro. Unlike currently available nucleoside reverse transcriptase (RT) inhibitors such as 3'-azido-3'-deoxythymidine (AZT), which have the 2',3'-dideoxy configuration and thereby cause DNA chain termination in the elongating proviral DNA, 4'SdNs do retain the 3'-alpha-OH moiety but also appear to work against retrovirus as proviral DNA chain terminators. Several 4'SdNs have been shown to be active against various laboratory and clinical HIV-1 strains including known drug-resistant HIV-1 variants. Among such 4'SdNs is 4'-azido-2'-deoxythymidine (4'-AZT) which exerts potent antiviral activity against wild type and AZT-resistant clinical HIV strains. More anti-HIV 4'SdNs have recently been reported including 4'-ethynylthymidine, 4'-ethynyl-2'-deoxy-D-ribofuranosyl-2,6-diaminopurine (4'-E-dDAP), 4'-ethynyl-2'-deoxyguanosine (4'-E-dG) and 4'-ethynyl-2'-deoxyadenosine (4'-E-dA). The latter three analogues are highly potent against HIV-1 and HIV-2 with EC50 values ranging from 0.0003 to 0.01 microM and have favorable cytotoxicity profiles with selective index (SI) values ranging from 975 to 2600. These 4'-ethynyl-2'-deoxynucleosides also exert potent activity against all known drug-resistant HIV-1 variants including the multi-dideoxynucleoside-resistant HIV and the variants with the 6-base pair inserts. Some of these compounds have favorable pharmacological properties and further development as potential therapeutics against HIV-1 infection is warranted.
Topics: Anti-HIV Agents; Deoxyribonucleotides; Drug Design; Drug Resistance, Viral; Drug Stability; HIV-1; Humans
PubMed: 12455229
DOI: 10.2174/1568005013343218 -
DNA Repair Dec 2007Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might...
Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H2O2, and the rpoB mutant frequencies were calculated. The frequency of the H2O2-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.
Topics: Adenosine Triphosphatases; Base Sequence; DNA Helicases; DNA, Bacterial; DNA-Binding Proteins; Deoxyribonucleotides; Escherichia coli Proteins; Mutation; Oxidation-Reduction; Oxidative Stress
PubMed: 17709303
DOI: 10.1016/j.dnarep.2007.06.013 -
Analytical Chemistry Oct 1988
Topics: Chromatography; Deoxyribonucleotides; Hydrogen-Ion Concentration; Methanol; Temperature
PubMed: 3239790
DOI: 10.1021/ac00170a024 -
Nucleic Acids Research Apr 2010Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA...
Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.
Topics: Cell Extracts; Cytidine Triphosphate; DNA Polymerase I; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Humans; Ribonucleotides; Taq Polymerase
PubMed: 20008099
DOI: 10.1093/nar/gkp1141