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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Linking number anomalies in DNA under conditions close to condensation. 265 31

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.
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PMID:Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria. 282 74

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

The inhibition of the semiconservative and restorative DNA synthesis caused by hyperthermia (30 to 60 min, 43 degrees C) was significantly higher in spleen cells than in thymus cells. The DNA repair synthesis of thymus cells measured at 37 degrees C was increased by about two times the initial value after a pre-incubation of 30 to 90 min and 30 to 60 min, respectively, with 37 and 43 degrees C, respectively. Under the same conditions, the 3H-thymidine incorporation into the DNA of spleen cells diminished proportionally to the pre-incubation time after a pre-incubation of 30 and 45 min, respectively, with 43 and 37 degrees C, respectively. When hyperthermia and inhibitors of DNA synthesis or DNA repair (hydroxyurea, 1-beta-D-arabinofuranosylcytosine, 3',5'-didesoxythymidine, and 3-aminobenzamide) were combined, overadditive effects--without cell specific particularities--were seen only in the case of 3-aminobenzamide. Only in thymus cells, the inhibitor of DNA topoisomerase II novobiocin caused an overadditive reinforcement of the inhibition induced by hyperthermia of the semiconservative DNA synthesis. The stimulation of DNA repair synthesis in thymus cells caused by novobiocin with the aid of DNA polymerase beta could be compensated by hyperthermia. The sedimentation of thymus and spleen cell nucleoids was increased after hyperthermia. The results suggest a special importance of DNA topology and of the DNA polymerase beta activity for the cellular effect of hyperthermia.
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PMID:[Deoxyribonucleic acid synthesis by rat thymus and spleen cells in vitro following hyperthermia]. 283 58

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


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