Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.
...
PMID:Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli. 254 33

Extensive digestion of the covalent intermediate between DNA and Saccharomyces cerevisiae DNA topoisomerase I with trypsin yields a 7-amino acid peptide covalently linked to DNA. Direct sequencing of the DNA-linked peptide identifies Tyr-727 as the active site tyrosine that forms an O4-phosphotyrosine bond with DNA when the enzyme cleaves a DNA phosphodiester bond. Site-directed mutagenesis of the cloned yeast TOP1 gene encoding the enzyme confirms the essentiality of Tyr-727 for the relaxation of supercoiled DNA by the enzyme. From amino acid sequence homology, Tyr-771 and -773 are readily identified as the active site tyrosines of Schizosaccharomyces pombe and human DNA topoisomerase I, respectively. Sequence comparison and site-directed mutagenesis also implicate Tyr-274 of vaccinia virus DNA topoisomerase as the active site residue. There appears to be a 70-amino acid domain near the carboxyl terminus of eukaryotic DNA topoisomerase I and vaccinia topoisomerase, within which the active site tyrosine resides.
...
PMID:Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I. 254 38

Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.
...
PMID:Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I. 255 29

Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2). The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000. The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome. This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.
...
PMID:Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. 282 64

The antiviral activity of ofloxacin, a new quinolone derivative, against vaccinia virus (VV), herpes simplex virus (HSV) and influenza virus (InfV) was evaluated in both in vitro and in vivo experiments. As a result, ofloxacin showed inhibitory activity against VV in cultured mammalian cells, and prevented formation of pox tail lesions in VV-infected mice. However, it was less effective against HSV and InfV than VV. The antiviral activity of ofloxacin assessed by VV tail-lesion test was strongest when administered to mice through the oral route daily for five consecutive days post-infection. Nalidixic acid and novobiocin, well-known gyrase inhibitors, showed only weak antiviral activity in both in vitro and in vivo tests against VV. It was also demonstrated that ofloxacin inhibited virus-specific DNA and RNA syntheses. It was more inhibitory to VV topoisomerase than cellular topoisomerases. Thus, ofloxacin has selectivity for VV.
...
PMID:Antiviral activity and inhibition of topoisomerase by ofloxacin, a new quinolone derivative. 282 66

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.
...
PMID:Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. 284 43

A soluble extract from purified vaccinia virus particles has been developed which displays site-specific initiation of transcription on exogenous DNA templates that carry cloned vaccinia virus early gene sequences. Bacterial plasmid vectors with segments of a strongly expressed early region of the vaccinia virus genome were active templates, whether in supercoiled or linear, truncated forms. Correct initiation, corresponding to that found in vivo, was observed for all early genes tested. The involvement of other factors besides the viral RNA polymerase was demonstrated by the loss of specific initiation upon partial purification of the enzyme. Initiation activity was restored by reconstitution of the system with factors lacking polymerase activity. The soluble system retained properties of transcription characteristic of intact viral cores, including (i) similar relative rates of initiation of various genes, (ii) multiple requirement for ATP, (iii) methylation and polyadenylation of transcripts, and (iv) inhibition by a topoisomerase antagonist.
...
PMID:A soluble transcription system derived from purified vaccinia virions. 298 38

The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.
...
PMID:Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus. 300 75

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
...
PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

A DNA topoisomerase has been purified from vaccinia virus cores. The native enzyme is composed of a single subunit of 32,000 daltons. The enzyme relaxes both positively and negatively supercoiled DNA in the absence of an energy cofactor. Enzymatic activity is stimulated by magnesium ions and inhibited by ATP, and during relaxation the topoisomerase changes the linking number of the DNA substrate by steps of one. Trapping of the covalent DNA-enzyme intermediate has been achieved, and analysis of the cleavage of duplex DNA by the enzyme reveals that it makes a single-strand break and forms a covalent bond through the 3'-end of the broken strand. Enzymatic activity and formation of the trapped intermediate are inhibited by the drugs novobiocin, coumermycin A1, and berenil. The virally encapsidated enzyme has novel properties but most closely resembles a eucaryotic type I topoisomerase.
...
PMID:Vaccinia virus encapsidates a novel topoisomerase with the properties of a eucaryotic type I enzyme. 303 53


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---