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 topoisomerases. 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 the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT.
Department of Cell Biology, Cornell University Medical College, New York, New York.
N
N
Klemperer
D J
DJ
Lyttle
D
D
Tauzin
P
P
Traktman
A J
AJ
Robinson
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009923:Orf virus / Q000201:enzymology*; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D014616:Vaccinia virus / Q000201:enzymology
D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I