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)

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

Nuclear novobiocin binding proteins (NBPs) from a set of mouse L cells have been extensively purified by affinity chromatography on novobiocin-Sepharose columns. The NBPs, specifically eluted with 100 micrograms of novobiocin per ml, exhibited equivalent DNA topoisomerase activities (measured as ATP-dependent relaxation or catenation of phi X174 replicative-form I DNA substrate) when extracted from equal numbers of wild-type (WT-4) mouse L cells growing logarithmically at 34 degrees C or at 38.5 degrees C, from ts A1S9 cells similarly cultivated at the low, permissive temperature or from revertant ts+ AR cells in exponential growth at either temperature. The NBPs isolated from similar numbers of ts A1S9 cells grown to midlogarithmic phase and then incubated for 24 hr at 38.5 degrees C (the nonpermissive temperature) showed no topoisomerase II activity. Preliminary NaDodSO4/polyacrylamide gel electrophoretic analysis of enzymatically active material revealed that the NBPs of WT-4 and ts+ AR cells grown at 34 degrees C comprised three major polypeptides of 76,000, 74,000, and 30,000 daltons and a number of larger molecular mass components present in trace amounts. The NBP of ts A1S9 cells grown at the permissive temperature was similar, except that the 30,000-kilodalton polypeptide was not detected. Such enzymatically active NBPs from WT-4 and ts+ AR cells were unaffected by 100 micrograms of novobiocin per ml, whereas the analogous preparation from ts A1S9 cells was totally inhibited. On the basis of these and other considerations, it is postulated that the ts A1S9 locus of mouse L cells encodes a temperature-sensitive polypeptide that is required for normal DNA topoisomerase II activity.
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PMID:ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity. 630 35

A type I DNA topoisomerase has been isolated from the nuclei of the flagellate Trypanosoma cruzi, using poly(ethylene glycol) fractionation and chromatography on hydroxyapatite and on phosphocellulose. The relaxation activity was ATP-independent, enhanced by Mg2+ and spermidine. The enzyme removed supercoils from negative and positive superhelical DNAs. Topoisomerase activity was associated with a polypeptide of Mr about 65000 as shown by glycerol gradient centrifugation and by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels.
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PMID:A type I DNA topoisomerase from Trypanosoma cruzi. 630 14

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.
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PMID:Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme. 632 14

The binding of DNA topoisomerase III (Topo III) to a single-stranded DNA substrate containing a strong cleavage site has been examined. The minimal substrate requirement for Topo III-catalyzed cleavage has been determined to consist of 7 bases; 6 bases 5' to the cleavage site and only 1 base 3' to the site. Nuclease P1 protection experiments indicate that the enzyme also binds to its substrate asymmetrically, protecting approximately 12 bases 5' to the cleavage site and only 2 bases 3' to the cleavage site. A catalytically inactive mutant of Topo III shows the same protection pattern as the active polypeptide, indicating that Topo III is a site-specific binding protein as well as a topoisomerase. Consistent with this view, an oligonucleotide containing a cleavage site is a more effective inhibitor and is bound more efficiently by Topo III than an oligonucleotide without a cleavage site.
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PMID:Escherichia coli DNA topoisomerase III is a site-specific DNA binding protein that binds asymmetrically to its cleavage site. 755 40

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.
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PMID:Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle. 770 2

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.
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PMID:Proteolytic footprinting of vaccinia topoisomerase bound to DNA. 774 4

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.
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PMID:Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta. 779 75

Unicellular Dinoflagellates represent the only eukaryotic Phylum lacking histones and nucleosomes. To investigate whether Dinoflagellates do have a nuclear matrix that would modulate the supramolecular organization of their non-nucleosomal DNA and chromosomes, cells of the free-living unarmored Dinoflagellate Amphidinium carterae were encapsulated in agarose microbeads and submitted to sequential extraction with non-ionic detergents, nucleases and 2 M NaCl. Our results demonstrate that this species has a residual nuclear matrix similar to that of vertebrates and higher plants. The cytoskeleton-nuclear matrix complex of A. carterae shows a relatively intricate polypeptide pattern. Immunoblots with different antibodies reveal several intermediate filament types of proteins, one of which is immunologically related to vertebrate lamins, confirming that these proteins are ancestral members of the IF family, which is highly conserved in eukaryotes. A topoisomerase II homologue has also been identified in the nuclear matrix, suggesting that these structures could play a role in organizing the Dinoflagellate DNA in loop domains. Taken together our results demonstrate that the nuclear matrix is an early acquisition of the eukaryotic nucleus, independent of histones and nucleosomes in such a way that the mechanisms controlling the two levels of organization in eukaryotic chromatin would be molecularly and evolutionarily independent.
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PMID:Dinoflagellates have a eukaryotic nuclear matrix with lamin-like proteins and topoisomerase II. 787 53

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
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PMID:Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis. 787 55


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