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)

In order to elucidate the mechanism by which the intercalative antineoplastic drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilizes the covalent topoisomerase II-DNA cleavage complex, the effect of the drug on the DNA cleavage/religation reaction of the type II enzyme from Drosophila melanogaster was examined. At a concentration of 60 microM, m-AMSA enhanced topoisomerase II mediated double-stranded DNA breakage approximately 5-fold. Drug-induced stabilization of the enzyme-DNA cleavage complex was readily reversed by the addition of EDTA or salt. When a DNA religation assay was utilized, m-AMSA was found to inhibit the topoisomerase II mediated rejoining of cleaved DNA approximately 3.5-fold. This result is similar to that previously reported for the effects of etoposide on the activity of the Drosophila enzyme [Osheroff, N. (1989) Biochemistry 28, 6157-6160]. Thus, it appears that structurally disparate classes of topoisomerase II targeted antineoplastic drugs stabilize the enzyme's DNA cleavage complex primarily by interfering with the ability of topoisomerase II to religate DNA.
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PMID:Stabilization of the topoisomerase II-DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide. 215 23

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA. 216 74

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.
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PMID:Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. 217 Mar 98

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate. 217 49

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

We have identified a nuclear protein which binds specifically to the first intron of rat alpha 2-macroglobulin gene. The protein became insoluble at low salt concentration retaining the binding specificity. Its molecular weight was estimated to be 22 kilodalton by a protein blotting procedure. The binding site of the protein determined by DNase I footprinting was an AT-strech which shared 80% homology with the cleavage consensus of Drosophila topoisomerase II.
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PMID:A nuclear factor which interacts with an AT-cluster in the first intron of rat alpha 2-macroglobulin gene. 244 39

The cytotoxic alkaloid, camptothecin, does not inhibit the nicking-closing activity of the wheat germ type I topoisomerase (topo I). However, consistent with a previous report on the Hela cell topo I (Hsiang, Y.-H., Hertzberg, R., Hecht, S., and Liu, L.F. (1985) J. Biol. Chem. 260, 14873-14878), the drug does enhance DNA breakage when enzyme reactions are terminated with SDS. Drug-enhanced breakage was observed over the range of salt concentrations where the enzyme is most active (25-200 mM monovalent cation). The presence of the drug did not appear to make the enzyme more processive in the range of salt concentrations from 100 to 170 mM, indicating that it probably does not affect the binding of the enzyme to DNA. Addition of high salt (0.5 M) to enzyme reactions containing camptothecin, prior to the addition of the detergent, prevented some, but not all of the drug-enhanced breakage. This result indicates that the drug causes some permanent, salt-stable nicking of the DNA, an observation that may explain its cytotoxic effects. A comparison of the breakage specificity in the presence of the drug with the consensus sequence for breakage determined previously (Been, M.D., Burgess, R.R., and Champoux, J.J. (1984) Nucleic Acids Res. 12, 3097-3114) indicated that the drug has a minimal effect on the sequence specificity of the enzyme. However, the drug enhanced breakage at different sites to quite different extents. Therefore, camptothecin should be useful for localizing topo I break sites in vivo, but quantitative comparisons on the relative frequencies of breakage at different locations should be avoided.
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PMID:The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase. 253 12

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.
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PMID:Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II. 254 64

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line. 255 59

The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.
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PMID:Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate. 255 67


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