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)

Pentamidine, diminazene aceturate (Berenil), isometamidium chloride (Samorin), and ethidium bromide, which are important antitrypanosomal drugs, promote linearization of Trypanosoma equiperdum minicircle DNA (the principal component of kinetoplast DNA, the mitochondrial DNA in these parasites). This effect occurs at therapeutically relevant concentrations. The linearized minicircles are protease sensitive and are not digested by lambda exonuclease (a 5' to 3' exonuclease), indicating that the break is double stranded and that protein is bound to both 5' ends of the molecule. The cleavage sites map to discrete positions in the minicircle sequence, and the cleavage pattern varies with different drugs. These findings are characteristic for type II topoisomerase inhibitors, and they mimic the effects of the antitumor drug etoposide (VP16-213, a semisynthetic podophyllotoxin analog) on T. equiperdum minicircles. However, the antitrypanosomal drugs differ dramatically from etoposide in that they do not promote detectable formation of nuclear DNA-protein complexes or of strand breaks in nuclear DNA. Selective inhibition of a mitochondrial type II topoisomerase may explain why these antitrypanosomal drugs preferentially disrupt mitochondrial DNA structure and generate dyskinetoplastic trypanosomes (which lack mitochondrial DNA).
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PMID:Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. 215 80

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
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PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52

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

The relationship between gene expression and the patterns of histone methylation in Drosophila cells has been investigated using inhibitors of transcription acting at various levels. Inhibition of ribosomal RNA synthesis and processing by 5-fluorouridine or of general RNA synthesis by camptothecin, an inhibitor of topoisomerase I, does not affect the methylation pattern of core histones. This suggests that the arrest of transcription per se is not involved in the changes in histone methylation such as those encountered in heat-shocked cells. However, ethidium bromide and novobiocin, which are known to disrupt nucleosome structure, and VM-26 (teniposide), a specific inhibitor of topoisomerase II, induce changes in histone methylation patterns which, though less severe, are similar to those observed under cellular stress. These results suggest that chromatin conformation is probably an important factor in the accessibility of histones to methyltransferases.
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PMID:Transcriptional inhibitors affecting topoisomerase II induce changes in histone methylation patterns similar to those induced by heat shock. 254 90

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.
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PMID:Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays. 255 90

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.
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PMID:DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators. 281 25

Supercoiled enriched PM-2 DNA has been relaxed by treating with calf thymus topoisomerase I and used in the preparation of a family of n-butylamine adducts of varying levels of substitution. The amine is cross-linked by formaldehyde to the exocyclic amino group of G when the DNA is in duplex form. These amine adducts of covalently closed relaxed (ccr) DNA, freed of the formaldehyde and n-butylamine reactants, have circular dichroism (CD) spectral properties similar to those previously reported for the adducts of calf thymus DNA [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987-4995]. In both instances, the CD transformation effected by increasing levels of substituted cationic amine is similar to that induced by solvents of high electrolyte content. The adducts also exhibit greatly increased electrophoretic mobility compared to unreacted controls or a control treated only with formaldehyde. Mobility changes in the presence of variable amounts of ethidium bromide demonstrate that this phenomenon is attributable to the formation of negative supercoils and is not due to denaturation or unwinding of the duplex. Incremental increases in superhelicity due to the attachment of the amine have been measured by reference to a topoisomerase ladder of underivatized PM-2 DNA and converted to changes in winding angle. As the extent of substitution increases, the rotational strength of the positive band above 260 nm decreases, and the winding angle increases in the nonlinear manner observed previously for underivatized PM-2 DNA [Baase, W. A., & Johnson, W. C., Jr. (1979) Nucleic Acids Res. 6, 797-814]. In fact, the relationship between these two properties is the same for both the adducts and the underivatized ccr species. Thus, the attachment of the amine has the same conformational effects as the electrolyte content of the solvent. The effect can be rationalized in terms of the reduction of the electrostatic free energy of the duplex due to site-bound or localized cation binding in the minor groove.
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PMID:Effects of charge modification on the helical period of duplex DNA. 284 28

A general, unrefined mechanism of type I DNA topoisomerase action involves several steps including DNA binding, single-strand scission, strand passage resealing, and, possibly, readoption of an active enzyme conformation. None of these steps requires an energy cofactor; however, we have shown previously that several mammalian type I topoisomerases are, in fact, inhibited by ATP. In this study, we wanted to examine which steps in the gross topoisomerase mechanism were sensitive or insensitive to ATP. Nitrocellulose filter binding experiments showed that ATP did not interfere with the binding of DNA by the enzyme and that ATP binding by topoisomerase was 5-fold greater in the presence of DNA than in its absence. Agarose gel electrophoresis in the presence or absence of ethidium bromide indicated that resealing was unaffected by added ATP. The addition of the adenine nucleotide did not alter the pattern of camptothecin-stimulated cleavage of DNA, indicating that strand scission was not the point of inhibition. To test whether strand passage or the readoption of an active conformation was an inhibited step, we used a unique DNA topoisomer as substrate. The results argued against readoption of an active enzyme conformation as an ATP-sensitive process.
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PMID:Mechanism of ATP inhibition of mammalian type I DNA topoisomerase: DNA binding, cleavage, and rejoining are insensitive to ATP. 284 29

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.
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PMID:Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles. 292 90

We have reported previously that rat liver mitochondria contain a topoisomerase and have shown it to be distinct from the nuclear enzyme by its sensitivity to Berenil and ethidium bromide. We report here some additional characterization. The enzyme differs further from its nuclear counterpart in its failure to bind to ssDNA cellulose and its chromatographic behavior on Sephadex; the latter procedure yields an Mr of 44 000 for the mitochondrial and 70 000 for the nuclear enzyme. The topoisomerase is strongly associated with mitochondrial membranes; only 10% of the activity could be extracted. The pH optimum of the enzyme falls between 6.0 and 8.5, with an NaCl optimum of 0.13 M in 0.1 M Tris (pH 8.3). Dithiothreitol is required, while N-ethylmaleimide is inhibitory. Tosylphenylalanine chloromethyl ketone, a serine proteinase inhibitor, abolishes activity; another, phenylmethanesulfonyl fluoride, has no effect. Berenil, a non-intercalating drug, and four of its analogues all inhibit with up to 100-fold differences in potency. No dependence on ATP, Mg2+, or both together could be shown. Neither novobiocin nor oxolinic acid shows any inhibitory effect. Nicked circles are generated in the presence of DMSO. These three observations are consistent with the topoisomerase being of the Type I class. Positively supercoiled pBR322 DNA, whose 6-8 positive turns were generated by altering solution conditions, is relaxed by the enzyme, indicating a lack of requirement for a negatively supercoiled substrate. We have also examined a partially purified preparation of the corresponding mitochondrial enzyme from mouse L cells. This enzyme is largely similar in properties to the rat liver enzyme. In isolated mitochondria, Berenil causes biphasic alterations in [3H]dATP incorporation into DNA, 10(-4) mM stimulating 2-fold, while higher concentrations inhibit. [3H]UTP incorporation into mitochondrial RNA also follows this pattern.
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PMID:Studies on mitochondrial type I topoisomerase and on its function. 298 52


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