PURIFICATION AND CHARACTERIZATION OF THE VACCINIA VIRUS DEOXYURIDINE TRIPHOSPHATASE EXPRESSED IN ESCHERICHIA-COLI

The deoxyuridine triphosphatase gene of vaccinia vi rus, encoded by th e open reading frame F2L, was cloned into Escherichia coli and express ed under the control of a bacteriophage T7 promoter. After induction o f T7 RNA polymerase by isopropyl beta-D-thiogalactopyranoside, a 16.5- kDa peptide accumulated to high levels. This 16.5-kDa protein was puri fied to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis rev ealed the enzyme to be a metalloenzyme; enzymatic activity is inhibite d by EDTA. This inhibition was reversed by the addition of Mg2+, Mn2+, or Zn2+. While the enzyme activity was highly specific for dUTP with an apparent K-m of 0.94 mu m, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a K-i of approximately 173 mu m. Also, protection studies demonstrated that nucleotide compet itors inhibit photoincorporation of the photoaffinity analogues [gamma -P-32]5-azido-dUTP and [gamma-P-32]8-azido-ATP. This suggests that whi le catalytic activity is limited to dUTP, other nucleotides can bind t he active site.