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)

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.
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PMID:Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro. 283 45

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

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.
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PMID:Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. 298 21

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.
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PMID:Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner. 298 7

We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme.
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PMID:Adenovirus infection elevates levels of cellular topoisomerase I. 298 7

This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single- and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.
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PMID:Inhibition of epipodophyllotoxin cytotoxicity by interference with topoisomerase-mediated DNA cleavage. 299 Apr 88

The effect of antitumor epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), on chromosomal DNA in mammalian cells was studied using SV40 virus-infected monkey cells as a model system. Treatment of SV40 virus-infected monkey cells with these drugs results in DNA breaks on intracellular SV40 DNA. The broken DNA strands are sensitive to phenol extraction, suggesting that they are associated with tightly linked protein(s). Several pieces of evidence suggest that DNA topoisomerase II is covalently linked to the broken SV40 DNA strands following drug treatment. ovobiocin, an inhibitor of topoisomerase II, blocks the epipodophyllotoxin-induced SV40 DNA breaks in vivo and in vitro. Epipodophyllotoxin-induced cleavage sites on intracellular SV40 DNA are strikingly similar to those produced on purified SV40 DNA by purified calf thymus DNA topoisomerase II. The protein-linked SV40 DNA is specifically immunoprecipitated by antisera against topoisomerase II. We thus conclude that epipodophyllotoxins induce chromosomal DNA breakage via DNA topoisomerase II. The physiological effects of epipodophyllotoxins on cell death, chromosomal DNA breakage, sister chromatid exchanges, and chromosomal aberrations may be the consequence of drug interaction with DNA topoisomerase II. Our present results are also consistent with the proposal that epipodophyllotoxins interfere with the breakage-reunion reaction of DNA topoisomerase II by stabilizing an enzyme-DNA complex in its putative cleavable state.
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PMID:Identification of DNA topoisomerase II as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. 299 63

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.
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PMID:Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation). 299 83

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.
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PMID:Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases. 300 Apr 18

Antitumor drugs from many chemical classes have been shown to induce protein-linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane-sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug-induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines.
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PMID:DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II. 300 16


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