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)

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.
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PMID:Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus. 164 1

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.
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PMID:Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. 165 Nov 2

We have introduced the novel application of a simple ethidium fluorescence assay, using covalently closed circular DNA, for the study of topoisomerase-targeted drugs. With the specificity of camptothecin for eukaryotic topoisomerases I and of VM26 for eukaryotic topoisomerases II, the two classes of enzymes can be assayed independently in crude extracts and during purification. These assays are fast, sensitive, and quantitative, have a large sample capacity, and eliminate the need for radioactive materials, filters, and agarose gels. We have demonstrated the use of this fluorescence assay to measure the inhibition of the relaxation and supercoiling activities of purified mammalian topoisomerases I and II and bacterial gyrase by nonintercalating drugs. Similarly, the production of drug-induced topoisomerase-mediated cleavable complexes was readily quantitated with both nonintercalating and intercalating drugs. When inhibition and cleavage with VM-26 were measured concurrently as a function of topoisomerase II concentration, a clear inverse relationship between topoisomerase II inhibition and cleavable complex production was observed. When the physiologically relevant salt K+L-glutamate- was used, quantitative relaxation by topoisomerase II was observed up to twice the salt concentration obtained with KCl. The enantiomer K+D-glutamate- gave exactly the same results, indicating that the enhancing role of glutamate- is non-stereospecific.
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PMID:Fluorometric assays for DNA topoisomerases and topoisomerase-targeted drugs: quantitation of catalytic activity and DNA cleavage. 165 89

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.
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PMID:Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae. 173 Jul 9

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
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PMID:Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation. 184 38

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.
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PMID:Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line. 184 51

The cleavage and religation reactions of eukaryotic topoisomerase II were studied by use of a 5'-recessed DNA substrate containing a strong recognition sequence for the enzyme. Cleavage of the DNA substrate was suicidal, that is the enzyme was unable to religate the cleaved DNA due to a release of DNA 5' to the cleavage position. With this substrate cleavage products accumulated with time in the absence of protein-denaturing agents, and the cleavage reaction was not reversible with salt. The suicide cleavage complexes contained a kinetically competent topoisomerase II enzyme as determined by the enzyme's ability to perform intermolecular ligation of the cleaved DNA to a free 3'-hydroxyl end on another DNA strand. The efficiency of the religation reaction depended on the ability of the religation substrate to base pair to the DNA in the cleaved enzyme-DNA complex. Higher levels of religation were obtained with dinucleotides than with long DNA substrates. Mononucleotides also were efficiently religated, indicating an ability of the enzyme to mediate religation without making contacts to a long stretch of nucleotides 5' to the cleavage position.
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PMID:Studies of the topoisomerase II-mediated cleavage and religation reactions by use of a suicidal double-stranded DNA substrate. 185 Nov 70

Terpentecin and clerocidin, microbial terpenoides, have been known to be potent antitumor antibiotics. However, the critical biochemical target of these terpenoides has not been identified. Our present studies, using purified mammalian topoisomerase II, have shown that terpentecin and clerocidin induce topoisomerase II-mediated DNA cleavage in vitro with comparable potency to that of demethylepipodophyllotoxin ethylidene-beta-D-glucoside. These terpenoides produced a similar DNA cleavage pattern which is distinctly different from those generated in the presence of the known topoisomerase poisons, demethylepipodophyllotoxin ethylidene-beta-D-glucoside and 4'-(9-acridinylamino)methanesulfon-m-anisidide. Brief heating at 65 degrees C, which abolishes completely the cleavable complex with demethylepipodophyllotoxin ethylidene-beta-D-glucoside, of the reaction mixture containing these terpenoides resulted in slight reduction in DNA cleavage. Thus, differently from other topoisomerase II-active antitumor agents, terpentecin and clerocidin induce formation of a cleavable complex which is stable for heat or salt treatments. The lack of significant DNA binding or intercalation activity of terpentecin and clerocidin suggests that topoisomerase II is a cellular target for these drugs.
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PMID:Induction of a heat-stable topoisomerase II-DNA cleavable complex by nonintercalative terpenoides, terpentecin and clerocidin. 185 67

We have previously shown that a cloned 480 bp DNA fragment that spans the 3'-enhancer region of the avian beta-globin gene cluster can become very tightly, perhaps covalently, bound to protein in avian nuclear matrices in vitro [Zenk et al. (1990) Biochemistry 29, 5221-5226]. This binding was not tissue-specific and was probably not mediated by topoisomerase enzymes. In the present study, we have examined avian nuclear matrices (or scaffolds) for the presence of very tight cellular DNA-protein complexes in the region of the beta-globin gene enhancer and of several other avian genes. Nuclear matrices were prepared by both high- and low-salt methods, and protein-DNA complexes were isolated by SDS/K+ precipitation after restriction enzyme digestion. In adult reticulocytes, up to 30% of the intact 3800 bp HindIII-EcoRI fragment that encompasses the beta-globin enhancer element may be very tightly bound to nuclear matrix protein. In adult avian thymus nuclei, the beta-globin enhancer is neither matrix-associated nor tightly bound to protein. In contrast, a 5.0-kb HindIII fragment of the malic enzyme gene is very tightly bound to nuclear matrix-associated protein in thymus cells, but not reticulocytes. The malic enzyme gene is active in thymus cells, and not in reticulocytes. These results suggests that certain regions of cellular DNA are very tightly, perhaps covalently, attached to nuclear matrix-associated proteins. Attachment follows a tissue-specific pattern that is associated with transcriptional activity.
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PMID:Avian nuclear matrix proteins bind very tightly to cellular DNA of the beta-globin gene enhancer in a tissue-specific fashion. 204 26

Two isoflavones, genistein (4',5,7-trihydroxyisoflavone) (1) and orobol (5,7,3',4'-tetrahydroxyisoflavone) (2) induced mammalian topoisomerase II dependent DNA cleavage in vitro. The cleavage activities of 1 and 2 were comparable to those of known antitumor agents with topoisomerase II dependent DNA cleavage activity such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP-16). Two flavones, fisetin (3,7,3',4'-tetrahydroxyflavone) (3) and quercetin (3,5,7,3',4'-pentahydroxyflavone) (4) showed topoisomerase II dependent DNA cleavage activity with similar potentials to that of Adriamycin. Addition of salt (0.5 M NaCl) to the reaction mixture containing genistein and topoisomerase II resulted in a great reduction of DNA cleavage, suggesting that the mechanism of the topoisomerase II dependent DNA cleavage induced by flavonoids is through the cleavable complex formation as seen with m-AMSA and VP-16. DNA unwinding assay using mammalian topoisomerase I showed that both 1 and 2 did not intercalate into DNA but both 3 and 4 intercalated like m-AMSA. Other structurally related flavonoids could not induce topoisomerase II dependent DNA cleavage, indicating that the restricted structures of flavonoids were required for the cleavage activity.
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PMID:Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol. 215 93


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