Faculty Collaboration Database

Identification and characterization of the orf virus type I topoisomerase. Virology 1995 Jan 10;206(1):203-15

Date

01/10/1995

Pubmed ID

7831775

DOI

10.1016/s0042-6822(95)80035-2

Scopus ID

2-s2.0-0028874237 (requires institutional sign-in at Scopus site)   27 Citations

Abstract

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.

Author List

Klemperer N, Lyttle DJ, Tauzin D, Traktman P, Robinson AJ

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Base Sequence
Cloning, Molecular
DNA Topoisomerases, Type I
DNA, Viral
Escherichia coli
Molecular Sequence Data
Orf virus
Point Mutation
Sequence Homology, Amino Acid
Vaccinia virus