pyrophosphate and deoxyuridine-triphosphate

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

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i). dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.

Topics: Animals; Cations; Deoxyuracil Nucleotides; Dimerization; Diphosphates; Escherichia coli; Hydrolysis; Kinetics; Leishmania major; Molecular Weight; Nucleotides; Phosphates; Phosphoric Monoester Hydrolases; Plastids; Pyrophosphatases; Recombinant Proteins; Sodium Fluoride; Substrate Specificity; Thermodynamics