pyrophosphate and deoxyguanosine-triphosphate

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (

Topics: Deoxyguanine Nucleotides; Deoxyguanosine; Diphosphates; DNA; DNA Polymerase beta; DNA-Directed DNA Polymerase; Humans; Kinetics; Substrate Specificity

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

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.

Topics: Base Pair Mismatch; Binding Sites; Catalysis; Cations; Crystallography, X-Ray; Deoxyguanine Nucleotides; Diphosphates; DNA-Directed DNA Polymerase; Kinetics; Magnesium; Manganese; Models, Molecular; Nucleotides; Protein Conformation

GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate. However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide-type intermediate analog. The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring. Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon-nitrogen bonds.

Topics: Amino Acid Sequence; Catalytic Domain; Deoxyguanine Nucleotides; Diphosphates; Escherichia coli; GTP Cyclohydrolase; Guanosine Triphosphate; Molecular Sequence Data; Mutagenesis, Site-Directed; Riboflavin; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Zinc

The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.

Topics: Bacterial Proteins; Deoxyguanine Nucleotides; Diphosphates; DNA Repair; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Isotope Labeling; Magnetic Resonance Spectroscopy; Oxygen Isotopes; Phosphoric Monoester Hydrolases; Pyrophosphatases; Water

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