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)

The effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.
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PMID:Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4'-(9-acridinylamino)methane-sulfon-m-anisidide. 131 38

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.
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PMID:DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases. 135

A mitochondrial type II DNA topoisomerase (topoIImt) has been purified to near homogeneity from the trypanosomatid Crithidia fasciculata. A rapid purification procedure has been developed based on the affinity of the enzyme for novobiocin, a competitive inhibitor of the ATP-binding moiety of type II topoisomerases. The purified enzyme is capable of ATP-dependent catenation and decatenation of kinetoplast DNA networks as well as catalyzing the relaxation of supercoiled DNA. topoIImt exists as a dimer of a 132-kDa polypeptide. Immunoblots of whole cell lysates show a single predominant band that comigrates with the 132-kDa polypeptide, indicating that the 264-kDa homodimer represents the intact form of the enzyme. Localization of the enzyme within the single mitochondrion of C. fasciculata (Melendy, T., Sheline, C., and Ray, D. S. (1988) Cell, in press) suggests an important role for topoIImt in kinetoplast DNA replication.
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PMID:Novobiocin affinity purification of a mitochondrial type II topoisomerase from the trypanosomatid Crithidia fasciculata. 253 75

The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.
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PMID:Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro. 298 12

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.
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PMID:DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization. 630 10

Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II alpha. This was selected using the procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure. The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L. A., and Perry, W. M. (1989) Mol. Endocrinol. 3, 603-604) as a possible core for leucine-zipper formation. Our experimental studies combine cross-linking and CD analysis. Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution. Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated alpha-helix. Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea. Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively. These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S. (1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis.
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PMID:A peptide fragment of human DNA topoisomerase II alpha forms a stable coiled-coil structure in solution. 761 54

The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.
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PMID:Nuclear distribution of Drosophila DNA topoisomerase II is sensitive to both RNase and DNase. 761 83

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.
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PMID:The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II. 816 75

We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 type II topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cleavage: four antitumor agents of different classes (an aminoacridine, a substituted anthraquinone, an ellipticine derivative and an epipodophyllotoxin) and one antibacterial quinolone. At eight nucleotide positions flanking the cleavage site, the same preferred bases were found regardless of which inhibitor was present. These preferred bases show dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner. In addition, we found that the preferred bases on the 5' side of each cleaved phosphodiester bond are highly specific to the inhibitor used in the cleavage reaction. These results strongly suggest that the inhibitors interact directly with the DNA bases at the cleavage site, placing the inhibitor binding site precisely at the site of DNA cleavage.
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PMID:Mutational analysis of a type II topoisomerase cleavage site: distinct requirements for enzyme and inhibitors. 838 18

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
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PMID:Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction. 866 52


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