pyrophosphate and 2--deoxycytidine-5--triphosphate

To investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine, as indicated by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3'-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat , suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol ) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester bond formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair.

Topics: Amino Acid Substitution; Catalytic Domain; Deoxycytosine Nucleotides; Diphosphates; DNA Adducts; DNA-Directed DNA Polymerase; Humans; Kinetics; Mutagenesis, Site-Directed; Protein Binding

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Hydrolysis; Nucleosides; Purine Nucleotides; Pyrimidine Nucleotides; Thymine Nucleotides

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Diphosphates; DNA; Luciferases; Luminescent Measurements; Models, Biological; Polymorphism, Single Nucleotide; Pyruvate, Orthophosphate Dikinase; ras Proteins; Reproducibility of Results; Thionucleotides; Thymine Nucleotides; Tumor Suppressor Protein p53

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.

Topics: Archaeal Proteins; Chromatography, Affinity; Cloning, Molecular; Codon; Databases, Nucleic Acid; Deoxycytosine Nucleotides; Deoxyuracil Nucleotides; Diphosphates; DNA-Directed DNA Polymerase; DNA, Archaeal; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli; Gene Expression; Genetic Vectors; Histidine; Hot Temperature; Oligopeptides; Polymerase Chain Reaction; Protein Denaturation; Pyrococcus; Pyrophosphatases; Recombinant Proteins; Sodium Chloride

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.

Topics: Base Sequence; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; DNA; DNA Polymerase I; Kinetics; Mathematics; Molecular Sequence Data; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Templates, Genetic

Topics: Bacteriophage phi X 174; Base Sequence; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; Diphosphates; DNA Replication; DNA-Directed DNA Polymerase; DNA, Viral; Intercalating Agents; Manganese; Mutation; Thymine Nucleotides

In connection with the characterization of two DNA polymerases (DPols) of Chlorella, we have extensively surveyed the literature on inhibition studies on DPols in various eukaryotes. By applying Tamiya's plot (1), we have obtained two parameters for each of the inhibitors, phi- and n-values, which express the enzyme sensitivity to the drug and the number of inhibitor molecules present in the enzyme-inhibitor complex that is principally involved in the inhibition, respectively. By inspecting these parameters for the three mammalian DPols, alpha-, beta-, and gamma-pols, as well as other eukaryotic DPols, we have found that: [1] inhibitors commonly utilized for characterizing various DPols can be classified into two major groups, each having two subgroups, on the basis of a comparison of the phi values among alpha-, beta-, and gamma-pols. Moreover, the grouping seems not to be merely coincidental, but to be intrinsically related to facets of the enzyme reaction, which may be taken to reflect evolutionary differences in DPol structure and function among the three DPols; [2] the remarkable n value, n = 1/2, that has been found for the inhibitors competitive with dCTP in Chlorella DPols has also been detected widely in many other eukaryotic DPols. Based on the first finding as well as many other data on various DPols, we have proposed an evolutionary scenario for eukaryotic DPols. Based on the second finding, we have hypothesized a novel role for dCTP as a cofactor, probably an apparent allosteric effector, in the nucleotide transfer reaction mechanism.

Topics: Aphidicolin; Arabinofuranosylcytosine Triphosphate; Biological Evolution; Cations, Divalent; Chlorophyta; Deoxycytosine Nucleotides; Dideoxynucleotides; Diphosphates; Diterpenes; Ethidium; Ethylmaleimide; Nucleic Acid Synthesis Inhibitors; Thymine Nucleotides