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 requirement for ATP hydrolysis in the initiation of RNA polymerase II (Pol II)-directed transcription and the relationship between ATP and novobiocin action led us to investigate whether novobiocin could inhibit transcription of the mouse metallothionein-I (MT-I) gene. Novobiocin inhibited the MT-I gene transcription in a fractionated rat hepatoma nuclear extract in a dose-dependent manner by direct interaction with a nuclear factor(s). This interaction prevented formation of stable preinitiation complexes but did not affect elongation of MT-I mRNA. Preincubation of the nuclear extract with ATP prevented the action of novobiocin on MT-I gene transcription. Although novobiocin is known to inhibit DNA topoisomerase II, VM-26, a specific inhibitor of this enzyme had no effect on the transcription. These results indicate that novobiocin blocks the Pol II-directed transcription by inhibiting formation of preinitiation complexes at an ATP-dependent step.
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PMID:Novobiocin inhibits initiation of RNA polymerase II-directed transcription of the mouse metallothionein-I gene independent of its effect on DNA topoisomerase II. 282 31

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.
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PMID:Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. 282 4

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.
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PMID:A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells. 282

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.
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PMID:Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria. 282 74

The effects of magnesium ions on interactions between Drosophila melanogaster topoisomerase II and its substrates were assessed by a number of kinetic and binding assays. Results indicated that the divalent cation plays two distinct functions in promoting enzyme-substrate interactions. One class of magnesium ions participates directly in enzyme-mediated DNA cleavage reactions. A second class of magnesium ions participates directly in topoisomerase II mediated ATPase reactions and functions by providing the enzyme with a magnesium-ATP substrate. In contrast, the divalent cation did not affect the quaternary structure of the enzyme, was not required for the site-specific binding of topoisomerase II to DNA, and did not affect the enzyme's ability to discern the topological state of its nucleic acid substrate.
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PMID:Role of the divalent cation in topoisomerase II mediated reactions. 282 26

An enzyme from Leishmania donovani that decatenates kinetoplast DNA networks into covalently closed monomeric circles has been isolated and characterized. The enzyme also relaxes supercoiled plasmid pBR322. The decatenation and relaxation reactions both require ATP and Mg2+. In both reactions the formation of topological isomers of unique linking numbers suggests that the enzyme is a type II DNA topoisomerase. Both the relaxation and decatenating activities are inhibited by novobiocin at a very high concentration.
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PMID:Decatenation of kinetoplast DNA by an ATP-dependent DNA topoisomerase from the kinetoplast hemoflagellate Leishmania donovani. 282 47

The effect of novobiocin, a coumarin class antibiotic, on rat ribosomal gene (rDNA) transcription in a fractionated extract derived from adenocarcinoma ascites cells (fraction DE-B) was studied. This drug inhibited transcription of rDNA by blocking initiation of transcription, whereas it had no effect on the elongation of the rRNA transcript. Order of addition experiments indicated that the novobiocin effect was at a step(s) in preinitiation complex formation. Preincubation of fraction DE-B with ATP before exposure to this antibiotic prevented inhibition of rDNA transcription. Since novobiocin has been shown to inhibit RNA polymerases II- and III-directed transcription of linear DNAs by interfering with a step(s) in the initiation reaction, these data suggest that initiation of transcription of all classes of RNA is inhibited by novobiocin by a mechanism independent of its effect on DNA topoisomerase II.
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PMID:Inhibition of RNA polymerase I-directed transcription by novobiocin. Potential use of novobiocin as a general inhibitor of eukaryotic transcription initiation. 283 4

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.
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PMID:Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro. 283 45

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.
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PMID:Topoisomerase-II-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA. 283 25

A topoisomerase capable of introducing positive supercoils into closed-circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135,000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65 degrees C to 100 degrees C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed-circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.
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PMID:Positive supercoiling catalysed in vitro by ATP-dependent topoisomerase from Desulfurococcus amylolyticus. 283 7


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