title: Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus.
abstract: We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10-20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.forename: Younguk
authors.initials: Y
authors.lastname: Sun
authors.forename: Mi
authors.initials: M
authors.lastname: Yu
authors.forename: Suk Hyung
authors.initials: SH
authors.lastname: Kwon
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001709:Biotechnology; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D019766:Genes, Archaeal; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction / Q000379:methods*; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase