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

Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.
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PMID:The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. 133 Mar 20

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.
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PMID:Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes. 165 20

Woodfruticosin (woodfordin C), a new cyclic dimeric hydrolyzable tannin having an inhibitory activity toward deoxyribonucleic acid (DNA) topoisomerase II, has been isolated from the leaves of Woodfordia fruticosa Kurz (Lythraceae) along with three known flavonol glycosides and three known flavonol glycoside gallates. The structure of wood fruticosin (woodfordin C) was determined by the use of two-dimensional nuclear magnetic resonance (2-D NMR) spectroscopy including heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC) techniques. Detailed analyses of the proton and carbon-13 NMR (1H- and 13C-NMR) spectra of six known flavonoids were performed.
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PMID:Constituents of the leaves of Woodfordia fruticosa Kurz. I. Isolation, structure, and proton and carbon-13 nuclear magnetic resonance signal assignments of woodfruticosin (woodfordin C), an inhibitor of deoxyribonucleic acid topoisomerase II. 196 10

DNA topoisomerase II was purified from calf thymus nuclei by a simple and fast four-step procedure: selective ammonium sulfate precipitation, chromatography on blue-Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on ATP or dATP, the Km values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5-8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaCl. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.
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PMID:Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa. 302 77

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.
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PMID:The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae. 608

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.
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PMID:Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites. 630 92

Nae I endonuclease must bind to two DNA sequences for cleavage. Examination of the amino acid sequence of Nae I uncovered similarity to the active site of human DNA ligase I, except for leucine 43 in Nae I instead of the lysine essential for ligase activity. Changing leucine 43 to lysine 43 (L43K) changed Nae I activity: Nae I-L43K relaxed supercoiled DNA to yield DNA topoisomers and recombined DNA to give dimeric molecules. Interruption of the reactions of Nae I and Nae I-L43K with DNA demonstrated transient protein-DNA covalent complexes. These findings imply coupled endonuclease and ligase domains and link Nae I endonuclease to the topoisomerase and recombinase protein families.
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PMID:DNA topoisomerase and recombinase activities in Nae I restriction endonuclease. 789 5

Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.
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PMID:Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage. 803 55

Replication of the Staphylococcus aureus plasmid pT181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, RepC. This oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. The resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified RepC, suggesting that the regulation of plasmid DNA replication requires post-replicational inactivation of the initiator protein as well as control of its synthesis.
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PMID:Replication-specific inactivation of the pT181 plasmid initiator protein. 823 21

Many tannins were previously identified as candidate topoisomerase poisons. Here we report further studies on sanguiin H-6, a dimeric ellagitannin isolated from Sanguisorba officinalis as an inhibitor of DNA topoisomerases. Catalytic strand-passing activities of topoisomerases I and II were inhibited in vitro with IC50 values of 1 microM and 0.01 microM, respectively. This inhibition was not associated with stabilization of covalent enzyme-DNA complexes but rather by a mechanism preventing formation of such covalent intermediates, as measured by interference with drug-induced cleavage in vitro. The IC50 values for topoisomerase I-DNA complexes induced by camptothecin and with topoisomerase II-DNA complexes induced by VP-16 were 0.02 microM and 0.16 microM, respectively. Pre-incubation studies followed by drug-dilution revealed that the in vitro inhibitory effects of sanguiin H-6 were irreversible, and for topoisomerase I, the test compound prevented enzyme-DNA interaction as seen by shifts in mobility on agarose gels. By measuring interference with drug-induced protein-linked DNA breaks in isolated HeLa nuclei, inhibition of topoisomerases I and II on a natural chromatin template was demonstrated with IC50 values of 5 microM and > 10 microM, respectively. Sanguiin H-6 inhibited HeLa cell growth with an ED50 of 12 microM and also interfered in a dose-dependent fashion with intracellular topoisomerase activities but with lower potencies than those observed using subcellular assay systems. Based on these studies, sanguiin H-6 could be broadly classified as a type of poison which does not stimulate the formation of cleavable-complexes, with intracellular activity but without any marked selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of DNA topoisomerases by sanguiin H-6, a cytotoxic dimeric ellagitannin from Sanguisorba officinalis. 839 Nov 44


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