Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Identification and characterization of the orf virus type I topoisomerase. 783 75

A 3.3-kb BamHI fragment from the center of the orf virus (OV) NZ2 genome has been sequenced, revealing three major open reading frames (ORFs) with homology to vaccinia virus (VAC) genes. These ORFs have been designated F2L, F3R, and F4R and the proteins they encode were found to be homologous to VAC genes H4L (RNA polymerase-associated protein RAP94), H5R (35-kDa virion envelope antigen) and H6R (topoisomerase), respectively. The OV ORFs are arranged on the genome in an almost identical manner to their VAC counterparts revealing the common evolutionary origin of the two viruses despite the extreme difference in their G+C content. Like its VAC counterpart, F3R was shown to be transcribed early and late during infection. S1 and primer extension analysis located the 5' ends of F3R early transcripts to a position 15-16 nt and 5-10 nt, respectively, downstream from an AT-rich sequence resembling a VAC early promoter. The 5' ends of F3R late transcripts were located to an A within the sequence 5'-TAAAG, 41 nt downstream from the early promoter and 17 nt upstream from the initiation codon. S1 analysis of F2L, which is transcribed only late in infection, revealed transcripts initiating from within the sequence 5'-TAAATG. No transcriptional start point could be detected for F4R but the VAC late transcriptional initiation sequence TAAAT was found close to the predicted translational start point. Another late promoter-like sequence, 5'-TAAATG, was found at the 3' end of F2L. This preceded a short ORF tentatively designated as F1L and predicted to be the beginning of a homologue of VAC H3L.
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PMID:Conservation of gene structure and arrangement between vaccinia virus and orf virus. 831 94

We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT decreases. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase (Kobs = 0.08 sec-1; Kcl = 0.22) are similar to those of the vaccinia virus enzyme.
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PMID:Characterization of a DNA topoisomerase encoded by Amsacta moore entomopoxvirus. 914 75