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Volume 271, Number 38, Issue of September 20, 1996 pp. 23506-23511
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Purification and Characterization of the Vaccinia Virus Deoxyuridine Triphosphatase Expressed in Escherichia coli

(Received for publication, May 22, 1996, and in revised form, July 15, 1996)

Nancy A. Roseman , Robert K. Evans ^¶ , Erica L. Mayer , M. Adrian Rossi and Mary B. Slabaugh ^''

From the Departments of  Biology and ^¶ Chemistry, Williams College, Williamstown, Massachusetts 01267 and the ^'' Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

The deoxyuridine triphosphatase gene of vaccinia virus, encoded by the open reading frame F2L, was cloned into Escherichia coli and expressed under the control of a bacteriophage T7 promoter. After induction of T7 RNA polymerase by isopropyl --thiogalactopyranoside, a 16.5-kDa peptide accumulated to high levels. This 16.5-kDa protein was purified to homogeneity and characterized. Gel filtration of the purified protein revealed a trimeric native structure. Biochemical analysis revealed the enzyme to be a metalloenzyme; enzymatic activity is inhibited 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 µ, inhibition studies show that 8-azido-ATP acted as a competitive inhibitor of dUTP with a K_i of approximately 173 µ. Also, protection studies demonstrated that nucleotide competitors inhibit photoincorporation of the photoaffinity analogues [-³²P]5-azido-dUTP and [-³²P]8-azido-ATP. This suggests that while catalytic activity is limited to dUTP, other nucleotides can bind the active site.

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