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.