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)

The ATP-dependent unknotting of phage P4 DNA is a highly specific assay for type II topoisomerases. Despite the unique specificity of the assay, however, its semiquantitative design has limited its use in studying the biochemical properties of these enzymes. To overcome this problem, we have modified the P4 DNA unknotting assay to provide a sensitive and reproducible method for quantifying topoisomerase II activity. Methods are described for accurate measurement of 10-100 ng of unknotted P4 DNA. Under the assay conditions employed, the initial rate of topoisomerase II activity was linear through 30 min. The quantitative assay has been used to determine biochemical and pharmacological parameters of purified topoisomerase II (p170). No topoisomerase II activity was observed in the absence of ATP; enzymatic activity was optimal between 0.5 and 1.0 mM ATP, but substrate inhibition occurred at concentrations above 1 mM. Eadie-Hofstee analysis with varying ATP concentrations gave an apparent Km for ATP of 0.24 mM and a maximal velocity under these conditions of 7.4 ng P4 DNA unknotted/min/ng topoisomerase II. IC50 values were determined for several topoisomerase inhibitors, including amsacrine, teniposide, and novobiocin. Inhibition by teniposide was found to be uncompetitive versus ATP, with a Ki of 3.7 microM. In contrast, inhibition by novobiocin was competitive versus ATP, indicating that teniposide and novobiocin inhibit topoisomerase II by different mechanisms.
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PMID:Quantitative adaptation of the bacteriophage P4 DNA unknotting assay for use in the biochemical and pharmacological characterization of topoisomerase II. 216 50

Novobiocin affects DNA metabolism in both prokaryotes and eukaryotes, resulting in cell death. In prokaryotes, the drug is a specific inhibitor of DNA gyrase, a type II topoisomerase that can be purified on a novobiocin-Sepharose column. The yeast type II topoisomerase is neither the biochemical, nor the genetic target of the antibiotic. We have purified the major yeast novobiocin binding proteins and identified one of them as the beta-subunit of the yeast mitochondrial F1 ATP synthetase, a protein highly conserved throughout evolution. The inactivation of this protein might explain the toxic effects of novobiocin on higher eukaryotic cells.
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PMID:The F1 ATP synthetase beta-subunit: a major yeast novobiocin binding protein. 217 64

In addition to its fundamental role of nucleating the formation of stable transcription complexes, the Xenopus laevis 5S RNA specific transcription factor, TFIIIA, promotes a variety of DNA-associated metabolic reactions. We report that TFIIIA can induce a DNA supercoiling catalyzed by the Xenopus laevis S-150 cell-free extract on plasmids containing a single copy of the Xenopus 5S RNA gene (somatic-type). Stimulated supercoiling occurs in the presence of high concentrations of ATP (4 mM) and at a factor to DNA ratio of 1 through a mechanism most likely involving type I topoisomerase. The highest level of stimulated supercoiling occurs when TFIIIA is incubated with DNA prior to the addition of the S-150 extract. Taken together, the experiments outlined in this report establish a reliable and seminal system in which TFIIIA-induced DNA supercoiling can be observed reproducibly.
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PMID:Reaction parameters of TFIIIA-induced supercoiling catalyzed by a Xenopus laevis cell-free extract. 231 14

Ovalbumin mRNA precursors were found to be almost quantitatively associated with the hen oviduct nuclear matrix. On the other hand, only one-third of the mature ovalbumin mRNA of whole nuclei was recovered in the nuclear matrix fraction. The binding of both the high molecular weight mRNA precursors and the mature-sized mRNA to the matrix displayed no difference in stability against salt, urea, or detergents. The mature mRNA, however, was found to be released selectively from the matrix by ATP. In contrast, the mRNA precursors remained completely bound to the nuclear substructure in the presence of ATP. Detachment of mRNA from the matrix also occurred in the presence of ADP, AMP plus pyrophosphate, or ATP analogs that contain nonhydrolyzable alpha, beta and beta, gamma bonds. Contrasting with the ATP-induced effect, addition of poly(A), ethidium bromide, or the copper chelator 1,10-phenanthroline to oviduct cell matrices caused an unspecific liberation of both mature and immature ovalbumin messengers. The release of the mature mRNA by ATP was found to be strongly inhibited by both nonintercalative and intercalative inhibitors of type II topoisomerase. These results suggest that the selection of the mature mRNAs for nucleocytoplasmic transport occurs at the release stage from the matrix (i.e. before translocation through the nuclear pore) and that reactions hitherto known to cause changes in the DNA secondary structure are associated with the detachment of mRNA from the nuclear substructure.
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PMID:Mature mRNA is selectively released from the nuclear matrix by an ATP/dATP-dependent mechanism sensitive to topoisomerase inhibitors. 243 4

Transcription of the Bombyx mori fibroin gene in a posterior silk gland extract can be separated into three functional steps on the basis of sensitivity to Sarkosyl: 1) formation of an initiation complex, which is blocked by 0.025% Sarkosyl; 2) conversion of the initiation complex to an elongation complex, a step sensitive to 0.05% Sarkosyl; 3) the subsequent elongation of RNA chain which occurs in the presence of 0.05% Sarkosyl. Whereas the last two steps are rapid and unaffected by template topology, the first step is slow and affected by DNA conformation. In the posterior silk gland extract, closed circular DNA forms a superhelical state and supports more rapid assembly of the initiation complex than linear DNA does. Both DNA supercoiling and rapid assembly of the initiation complex require ATP and are abolished by the addition of a topoisomerase II inhibitor VP16. These results suggest that DNA supercoiling enhances the fibroin gene transcription by facilitating formation of the initiation complex.
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PMID:DNA supercoiling facilitates formation of the transcription initiation complex on the fibroin gene promoter. 245 21

The role of topoisomerase enzymes in the response of HeLa S3 cells to ionizing radiation was investigated. Exposure of cells to 100 Gy of X-radiation had no detectable effect either on the total cellular topoisomerase activity as measured by the relaxation of supercoiled plasmid DNA by cell sonicates or on the total cellular topoisomerase II activity as measured by plasmid DNA catenation. Total topoisomerase II activity remained constant for up to 90 min after cell irradiation. The effect of 2 drugs (caffeine and novobiocin) which inhibit topoisomerase II activity on the HeLa cell response to radiation was determined. Both drugs were found to inhibit topoisomerase II in vitro and to inhibit the recovery of nucleoid sedimentation in irradiated cells in vivo to the same extent. Topoisomerase II was inhibited by 50% by exposure to 10 mM caffeine and 0.79 mM novobiocin. At low concentrations neither drug affected the induction frequency, nor the rejoining rate, of DNA double-strand breaks. Caffeine (5 mM) inhibited the short-term recovery of cells from radiation while novobiocin (0.79 mM) had no detectable effect on the capacity of cells to recover from radiation exposure. The results indicate that topoisomerase II is not required for DNA double-strand break rejoining though it could be required for the recovery of DNA coiling in the irradiated cell. If topoisomerase II is involved at all in cell recovery from irradiation, this role does not apparently involve an ATP-dependent enzyme activity.
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PMID:Topoisomerase activity in irradiated mammalian cells. 253 62

Circular plasmid DNA was efficiently converted into huge catenated intranuclear networks by incubation with isolated nuclei in the presence of ATP. The network production is abolished by omission of ATP, strongly inhibited by etoposide (VP-16), but only slightly inhibited by antibody to topoisomerase I, indicating that the major enzyme responsible for catenation is DNA topoisomerase II. Under optimal conditions, a single nucleus incorporates about 4.2 x 10(4) DNA rings into its networks. Under the light microscope, networks retrieved from nuclei appear like spheres of various sizes. Sedimentation analysis showed that most of the networks are composed of thousands of catenated rings, which was confirmed by electron microscopy. Data from experiments that caused partial disruption of the networks were submitted to analysis based on probable models of catenane structure. The results suggest that the predominant pattern is a linear alignment of catenated rings. Similar networks are formed when the nuclear scaffold is incubated with circular DNA in the presence of nuclear extract containing topoisomerase II. Titration experiments showed that the scaffold binds a stoichiometric amount of the substrate and that a critical level of DNA is required for network formation. The results are consistent with the idea that DNA-binding sites are fixed on the scaffold and mediate catenation of bound DNA circles by holding them in close proximity to each other. We propose that catenation by the nuclear scaffold also occurs in intact nuclei, suggesting additional roles for the scaffold in vivo.
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PMID:Incorporation of exogenous circular DNA into large catenated networks in isolated nuclei. Evidence for involvement of the nuclear scaffold. 254 Jan 99

DNA topoisomerases are complex and unique enzymes which alter the topological state of DNA without changing its chemical structure. Between the type I and II enzymes, topoisomerases carry out a multitude of reactions, including DNA binding, site specific DNA cleavage/religation, relaxation, catenation/decatenation, and knotting/unknotting of nucleic acid substrates, DNA strand transfer, and ATP hydrolysis. In vivo, topoisomerases are involved in many aspects of nucleic acid metabolism and play critical roles in maintaining chromosome and nuclear structure. Finally, these enzymes are of clinical relevance, as they appear to be the primary cellular targets for many varied classes of antineoplastic agents. Considering the importance of the topoisomerases, it is distressing that we know so little about their enzymatic mechanisms. Many major questions remain. Just a few include, "How do topoisomerases recognize their nucleic acid interaction sites?"; "What amino acid residues comprise the enzymes' active sites?"; "What are the conformational changes that accompany DNA strand passage?"; "How does phosphorylation stimulate enzyme activity?"; "How does topoisomerase function when it is part of an immobilized structure such as the nuclear matrix or the mitotic chromosome scaffold?"; and "How do antineoplastic agents interact with their topoisomerase targets and stabilize covalent enzyme.DNA cleavage products?" Clearly, before the physiological functions of the topoisomerases can be fully described, these and similar issues will have to be addressed. Hopefully, the next several years will produce answers for at least some of these important questions.
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PMID:Biochemical basis for the interactions of type I and type II topoisomerases with DNA. 254 Apr 96

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.
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PMID:In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone. 254 Sep 3

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.
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PMID:Studies on DNA polymerases and topoisomerases in archaebacteria. 254 77


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