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)

We have analyzed the long-range distribution of topoisomerase II-mediated cleavages induced in an amplified human c-MYC gene locus in the presence of several antitumor agents. The long-range cleavage patterns were found to be nonrandom and similar for all antitumor drugs tested. Cleavages occurred within several kilobase-long areas (approximately 5 kb) highly accessible to topoisomerase II and separated by extended regions (approximately 70-100 kb) of less accessibility, possibly reflecting the mode of DNA organization into loops along the chromosome. Within the cleavage areas, the patterns of cleavage sites showed a certain dependence on the type of drug used for entrapment of topoisomerase II-DNA complexes. Importantly, distribution of cleavage areas in native chromatin and histone-depleted nuclei was very similar, if not identical, suggesting that the primary target of antitumor agents in vivo is topoisomerase II associated with the high-salt-insoluble nuclear matrix. These data show that matrix-attached DNA is preferentially damaged by topoisomerase II-targeting agents, which may be an important cellular event contributing to drug-induced cell death.
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PMID:Different topoisomerase II antitumor drugs direct similar specific long-range fragmentation of an amplified c-MYC gene locus in living cells and in high-salt-extracted nuclei. 781 96

Topoisomerase II overexpressed in yeast was purified to near homogeneity. The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological assays at different protein and salt conditions. First, sedimentation equilibrium was preferred over the other analytical ultracentifuge methods as it is based on firm theoretical grounds and does not require assumptions about the shape of the molecule. The tendency of topoisomerase II to self-associate into dimers was confirmed and shown to depend on both the enzyme concentration and the concentration of salt used. Analysis at five initial protein concentrations (from 0.08 to 1.05 mg/mL, i.e., 0.5-65 microM) provided evidence for a single monomer-dimer equilibrium characterized at 150 mM KCl and 20 degrees C by an association constant, Ka, of approximately 4.8 10(5) M-1 and a delta G degree of approximately -7.5 kcal mol-1. Under these conditions, for a topoisomerase II concentration of 0.08 mg/mL (i.e., 0.5 microM) in the ultracentrifuge cell, almost 80% of the enzyme were found dissociated. Increase of KCl (from 80 to 400 mM) in the medium provoked a continuous change of the association equilibrium so that a value of Ka approximately 10(5) M-1 corresponding to delta G degree approximately -7 kcal mol-1 was found for topoisomerase II in 400 mM KCl at 20 degrees C. Second, circular dichroism (CD) showed the sensitivity of the topoisomerase II secondary structure to salt concentration, the observed variations being apparently dependent upon the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions. 789 59

The rate of relaxation of supercoiled DNA by purified vaccinia topoisomerase I is stimulated 20-fold by 5 mM ATP. A similar effect is elicited by GTP, CTP, UTP, dATP, and adenosine 5'-(beta, gamma-imido)triphosphate. ATP-mediated rate enhancement requires salt as a coactivator. ADP and inorganic pyrophosphate also stimulate relaxation 10-20-fold, whereas AMP and inorganic phosphate have little effect. A model for allosteric activation of topoisomerase by nucleotides is suggested.
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PMID:Stimulation of vaccinia topoisomerase I by nucleoside triphosphates. 796 68

Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
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PMID:Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26. 798 Jun 48

Topoisomerase II alpha (170 kDa) expressed in human HL-60 cells is heterogeneous in charge. By two-dimensional electrophoresis and chromatofocussing two major subforms with pI of 6.5 and 6.7 can be resolved. By preparative anion-exchange chromatography we separated the known topoisomerase II isoenzymes (170/180 kDa) and in addition a late-eluting 170 kDa form, which has not been described before. The catalytic optimum of this late-eluting form is shifted to pH 9.4. It is more than 100-fold resistant to orthovanadate, amsacrine or etoposide, and has an increased salt stability. SDS-treatment induces covalent attachment of this enzyme fraction to calf thymus DNA in the absence of drug. The latter observations indicate an increase in DNA-binding. In the tightly DNA-bound state the late-eluting enzyme is not targeted by cleavable complex forming drugs. Accordingly, cells may become drug-resistant by expressing this form predominantly.
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PMID:Drug-sensitivity and DNA-binding of a subform of topoisomerase II alpha in resistant human HL-60 cells. 799 49

Several clinically important drugs utilized in cancer chemotherapy inhibit type I (Topotecan) or type II (amsacrine, etoposide) DNA topoisomerases by stabilizing the formation of DNA-topoisomerase complexes (topoisomerase-DNA cross-links). In various cell lines, the magnitude of drug-induced DNA-protein cross-link production correlates with the magnitude of cytotoxicity induced by the drugs. We developed a simple filter-binding assay that can measure drug-induced DNA-protein cross-links in leukemia cells obtained directly from patients because the assays most widely used for assessment of drug-induced DNA-protein cross-links in cells [sodium dodecyl sulfate (SDS)/KCl precipitation and alkaline elution] are not readily applicable for use on patient material. HL-60 human leukemia cells or freshly isolated patients' leukemia cells were incubated with Topotecan, etoposide, or amsacrine; lysed with SDS; and applied to nitrocellulose filters in a low-salt buffer. DNA is retained on the filter only if it is covalently bound to protein. The amount of DNA retained on the filter is quantified by hybridization to the alu sequence of DNA, which is distributed ubiquitously in the human genome. Using radiolabeled cells, we compared the filter-binding assay directly with the SDS/KCl precipitation assay in the detection of etoposide- or amsacrine-induced DNA-protein cross-links in HL-60 cells and amsacrine-resistant HL-60/AMSA cells. Both the SDS/KCl precipitation assay and the filter-binding assay detected etoposide-induced DNA-protein cross-links in HL-60 and HL-60/AMSA cells and detected a greater frequency of amsacrine-induced DNA-protein cross-links in HL-60 cells than in HL-60/AMSA cells. The filter-binding assay detected DNA-protein cross-links in freshly isolated leukemia cells exposed to Topotecan in vitro. The ratios of DNA retention for Topotecan-treated versus untreated cells from leukemia patients ranged from 1.8 to 11.5. The heterogeneity of this detected cross-linking was as might be expected if the assay were predictive of the antileukemic action of Topotecan, which is variable. This new filter-binding technique may be useful for predicting the sensitivity of individual patients' tumors to drugs that inhibit type I or type II DNA topoisomerases.
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PMID:Quantification of topoisomerase-DNA complexes in leukemia cells from patients undergoing therapy with a topoisomerase-directed agent. 800 59

Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from gamma-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. In contrast, only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, as shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining). This difference was thought to be due to the increased accessibility of SQ-9G DNA in vitro. We suggest that a looser association of SQ-9G DNA with the nuclear matrix both promotes DNase I digestion and affects the ability of SQ-9G nucleoids to maintain positive DNA supercoils after irradiation. These data implicate the DNA matrix attachment region in the expression of radiation sensitivity in the cell lines studied.
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PMID:A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines. 809 63

The mechanism of inhibition of eukaryotic DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] by a member of the bisdioxopiperazine family of anticancer drugs, ICRF-193, was investigated by using purified yeast DNA topoisomerase II. In the absence of ATP, ICRF-193 has little effect on the binding of the enzyme to various forms of DNA. In the presence of ATP, the drug converts the enzyme to a form incapable of binding circular DNA. Incubation of a preformed circular DNA-enzyme complex with ICRF-193 and ATP converts the complex to a form stable in molar concentrations of salt. These results can be interpreted in terms of the ATP-modulated protein-clamp model of type II DNA topoisomerases [Roca, J. & Wang, J. C. (1992) Cell 71, 833-840]; ICRF-193 can bind to the closed-clamp form of the enzyme and prevents its conversion to the open-clamp form. This interpretation is further supported by the finding that whereas both ATP and the drug are needed to form the salt-stable circular DNA-enzyme complex, ATP is not needed for maintaining this complex; furthermore, a signature of the closed-clamp form of the enzyme, Staphylococcus aureus strain V8 endoproteinase cleavage site at Glu-680, is observed if the enzyme is incubated with both ATP and ICRF-193. Inhibition of interconversion between the open- and closed-clamp forms of type II DNA topoisomerases offers a new mechanism in the selection and design of therapeutics targeting this class of enzymes.
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PMID:Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. 812 81

A type 1 DNA topoisomerase has been purified from the nuclei of the kinetoplast hemoflagellate Leishmania donovani using polyethylene glycol fractionation and chromatography on hydroxylapatite, phosphocellulose and phenylsepharose column. The relaxation activity is ATP independent. Mg2+ is an essential cofactor for the reaction with an optimum at 10 mM. Mg2+ can be substituted by Mn2+ at 5 mM concentration. The relaxation reaction exhibits a salt optimum at 100 mM KCl. The enzyme can not remove supercoils from positive superhelical DNAs nor can induce supercoiling of relaxed DNAs. The topoisomerase activity is associated with a polypeptide of molecular weight about 67 kDa as shown by sephacryl-S200 gel filtration and by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels.
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PMID:A type 1 DNA topoisomerase from the kinetoplast hemoflagellate Leishmania donovani. 814 68

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


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