Authors: Y Cho H S Lee Y J Kim S G Kang SJ Kim JH Lee
Publish Date: 2007/06/05
Volume: 9, Issue: 4, Pages: 450-458
Abstract
Genomic analysis of the hyperthermophilic archaeon Thermococcus onnurineus NA1 TNA1 revealed the presence of a 471bp open reading frame with 93 similarity to the dUTPase from Pyrococcus furiosus The dUTPaseencoding gene was cloned and expressed in Escherichia coli The purified protein hydrolyzed dUTP at about a 10fold higher rate than dCTP The protein behaved as a dimer in gel filtration chromatography even though it contains five motifs that are conserved in all homotrimeric dUTPases The dUTPase showed optimum activity at 80°C and pH 80 and it was highly thermostable with a halflife t 1/2 of 170 min at 95°C The enzymatic activity of the dUTPase was largely unaffected by variations in MgCl2 KCl NH42SO4 and Triton X100 concentrations although it was reduced by bovine serum albumin Addition of the dUTPase to polymerase chain reactions PCRs run with TNA1 DNA polymerase significantly increased product yield overcoming the inhibitory effect of dUTP Further addition of the dUTPase allowed PCR amplification of targets up to 15 kb in length using TNA1 DNA polymerase This enzyme also improved the PCR efficiency of other archaeal family B type DNA polymerases including Pfu and KOD
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