호열성 dUTPase의 특성과 PCR 응용

Abstract

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1(TNA1) revealed the presence of an open reading frame consisting of 468 bp similar to dUTPases from Pyrococcus woesei and P. furiosus (83% identity and 93% similarity). TNA1 dUTPase has five motifs conserved in all dUTPases from eukarya and prokarya origins. The dUTPase gene was cloned and expressed in Escherichia coli. The purified protein behaved as a dimer in gel filtration and was able to hydrolyse both dUTP and dCTP. The optimum activity toward dUTP occurred at 80°C and pH 8.0. Moreover, the enzyme activity was highly dependent on Mg2+ concentration with optimum activity at 1mM to 5mM which is usually used at PCR amplification. TNA1 dUTPase activity was a little affected by KCl and (NH4)2SO4, while Tritoxn X-100 slightly increased dUTPase activity. The enzyme displayed thermostability with half-life (t1/2) of 170 min at 95°C. TNA1 dUTPase enhanced the PCR in a broader range of target lengths in combination with TNA1 DNA polymerase. Moreover, TNA1 dUTPase increased the yield of PCR products as much as other archaeal DNA polymerases.