IDENTIFICATION AND CHARACTERIZATION OF THE ORF VIRUS TYPE-I TOPOISOMERASE

Vaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively, encode type I DNA top oisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with t he VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The 545 protein was expressed in Escherichia coil under the control of an inducible T7 pro moter, partially purified, and shown to be a bona fide type I topoisom erase. Like the VV enzyme, the OV enzyme relaxed negatively supercoile d DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SD S-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PC R used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at ni 677 that was predicted t o replace a highly conserved Tyr residue with a Cys. This allele direc ted the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. In cubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an in distinguishable set of DNA cleavage fragments, although the relative a bundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV en zyme, we demonstrated that the OV enzyme also cleaved efficiently imme diately downstream of the sequence CCCTTdown arrow. (C) 1995 Academic Press, Inc.