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

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.
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PMID:Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. 796 97

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'-CCCTT decreases in the scissile strand. The protein-DNA interface entails essential contacts with four phosphate moieties within the CCCTT motif, including the scissile phosphate, and three phosphates within the GGGAA sequence on the noncleaved strand. Critical protein-phosphate contacts are arrayed across the minor groove of the DNA helix. Base-specific contacts with the pentamer element are within the major groove and are situated on the opposite face of the helix. Thus, vaccinia topoisomerase binds circumferentially to its target site in duplex DNA. This binding mode suggests that the eukaryotic enzyme adopts a toroidal shape in the DNA-bound state. Conformational isomerization of the bound protein provides a plausible mechanism for DNA relaxation.
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PMID:Vaccinia topoisomerase binds circumferentially to DNA. 798 46

A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.
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PMID:Protein footprinting by the combined use of reversible and irreversible lysine modifications. 799 55

When expression of the vaccinia virus gene encoding RAP94 (a protein that is associated with the viral multisubunit RNA polymerase and confers transcriptional specificity for early promoters) was repressed, the infectious virus yield was reduced by more than 99%. Nevertheless, intermediate- and late-stage viral gene expression and formation of ultrastructurally mature, membrane-enveloped virions occurred under the nonpermissive conditions. The RAP94-deficient particles contained the viral genome, structural proteins, early transcription factor, and certain enzymes but, unlike normal virions, had low or undetectable amounts of the viral RNA polymerase, capping enzyme/termination factor, poly(A) polymerase, DNA-dependent ATPase, RNA helicase, and topoisomerase. The presence of these viral enzymes in the cytoplasm indicated that RAP94 is required for targeting a complex of functionally related proteins involved in the biosynthesis of mRNA.
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PMID:Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein. 810 1

Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.
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PMID:Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex. 827 53

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

The ts16 mutation of vaccinia virus WR (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment. The I7 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C). Electron microscopy revealed a defect in virion assembly at 40 degrees C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core. The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.
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PMID:Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component. 838 72

Stabilization of crossings of pairs of DNA helices by binding of eukaryotic DNA topoisomerase II was studied by two types of experiments. In one, mixtures of yeast DNA topoisomerase II and supercoiled DNA were incubated with vaccinia virus topoisomerase, and the linking numbers of the DNA products were measured to quantitate supercoils that were constrained by the stoichiometrically bound yeast enzyme molecules; in parallel, the same yeast enzyme-supercoiled DNA mixtures were incubated with a nonhydrolyzable ATP analog AMPPNP (adenosine 5'-(beta, gamma-imido)triphosphate) instead of the vaccinia enzyme, and DNA linking number changes following the addition of AMPPNP were measured to monitor DNA transport mediated by the yeast enzyme and AMPPNP. In the second type of experiments, formation of knotted DNA rings by the addition of AMPPNP to mixtures of yeast DNA topoisomerase II and different topological forms of DNA rings was studied. These experiments indicate that binding of yeast DNA topoisomerase II to DNA crossings is significant, especially in low salt media containing Mg(II), and that this mode of binding strongly affects DNA knotting. It appears, however, that stabilization of DNA crossovers by the eukaryotic type II enzyme is not directly related to its DNA transport activity.
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PMID:On the simultaneous binding of eukaryotic DNA topoisomerase II to a pair of double-stranded DNA helices. 839 Sep 87

The 32-kDa topoisomerase I encoded by vaccinia virus relaxes supercoiled DNA in a manner which is mechanistically equivalent to that utilized by eucaryotic enzymes. Its amino acid sequence contains significant homology to the enzymes encoded by Saccharomyces cerevisiae, Saccharomyces pombe, human cells, and other poxviruses. The small size of the viral enzyme, and its essentiality in the viral life cycle, make it ideally suited for structural and functional analysis. In this report we present the construction and analysis of 15 mutant alleles of the topoisomerase containing amino acid substitutions in a highly conserved region. The enzymes encoded by these alleles were expressed in Escherichia coli and various parameters of their activity were examined. All of the alleles which show diminished (seven alleles) or abrogated (three alleles) DNA relaxation activity are deficient in DNA cleavage and the concomitant formation of the covalent enzyme/DNA intermediate. None are deficient in the prior step of noncovalent interaction with substrate DNA. Five of the mutant enzymes show significant temperature sensitivity in vitro. The extent of in vitro activity of the enzymes shows a good but incomplete correlation with the enzymes' abilities to lethally induce the resident lambda prophage within E. coli BL21(DE3) (via illegitimate recombination). Mutations in 1 amino acid, in particular, impair prophage induction in vivo more significantly than DNA relaxation in vitro. In sum, these studies suggest that this region of the topoisomerase (amino acids 216-225) plays a proximal role in mediating DNA cleavage and the covalent interaction between the 3'-phosphoryl of the nicked DNA and tyrosine 274 of the vaccinia topoisomerase I. The studies also provide useful reagents for the molecular genetic analysis of the role of the topoisomerase within the context of vaccinia virus infection.
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PMID:Biochemical analysis of mutant alleles of the vaccinia virus topoisomerase I carrying targeted substitutions in a highly conserved domain. 839 54

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.
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PMID:Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. 856 95


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