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)

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.
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PMID:A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer. 238 42

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

DNA topoisomerase I has been purified from homogenates of mature Xenopus laevis ovaries. The initial stages in purification of the native enzyme employed a rapid series of three chromatographic steps, followed by gel filtration performed in the presence of sodium dodecyl sulfate. Polypeptides that might represent topoisomerase I were identified by specific labeling of the topoisomerase species with radioactive DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of topoisomerase I radiolabeled with DNA identified three polypeptides with mobilities consistent with sizes of 165, 125, and 88 kDa. All three polypeptides were found to possess topoisomerase activity following elution from the gel and renaturation. Partial proteolytic digestion of the radiolabeled 165-, 125-, and 88-kDa polypeptides with Staphylococcus aureus V8 endoproteinase resulted in identical autoradiographic patterns. This suggests that the 125-kDa and 88-kDa polypeptides may be degradation products of the 165-kDa species. The 165-kDa topoisomerase I exhibited the same sensitivity to camptothecin as the total, native topoisomerase I fraction.
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PMID:A high molecular weight topoisomerase I from Xenopus laevis ovaries. 253 54

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.
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PMID:Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster. 254 46

Interactions of novobiocin (NB) and/or nalidixic acid (NA) with some cytotoxic agents--UV light (UV), X-rays, methylmethanesulfonate (MMS), bleomycin (BM), adriamycin (AM), cis-diaminedichloroplatinum(II) (CDDP), mitomycin C (MIT), ethidiumbromide (EB), and suramin (SA)--have been investigated in thymic (T) and splenic (S) cells of the rat in vitro by measuring semiconservative (SDS) and unscheduled (UDS) DNA synthesis as well as sedimentation and viscosity of nucleoids. Combining NB (900 micrograms/ml) and/or NA (1800-3600 micrograms/ml) with UV, MMS, BM, AM, MIT, and SA resulted in additive effects on SDS and UDS. Synergistic actions were observed in T- and S-cells simultaneously treated by NB and CDDP, whereas the effects of NB could be antagonized to some extent by EB and vice versa. In X-irradiated (greater than or equal to 28 Gy) cells pretreated by NB (NA), UDS was diminished (T-cells) or enhanced (S-cells). The results are consistent with the following postulates: 1 degree in S-cells, DNA is much more supercoiled than in T-cells but in the opposite sense (positive superhelicity). 2 degrees DNA supercoiling (DNA compactness) is influenced therefore by DNA-topoisomerase inhibitors and intercalating agents in a highly agent- and cell-specific manner.
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PMID:[Interactions of DNA topoisomerase inhibitors novobiocin and nalidixic acid with some cytotoxic agents. In vitro investigations in rat thymic and splenic cells]. 277 9

Sites of an endogenous activity that has the properties of a DNA topoisomerase I have been identified on the palindromic ribosomal RNA genes of the slime mould Dictyostelium discoideum. This was done in vitro, by treating isolated nuclei with sodium dodecyl sulphate, which denatures topoisomerase during its cycle of nicking, strand passing and resealing, and hence reveals the DNA cleavages. It was also done in vivo using the drug camptothecin, which is believed to stabilize the cleavable complex of topoisomerase I plus DNA, hence increasing the chances of cleavage when sodium dodecyl sulphate is subsequently added. The cleavages in vitro and in vivo were mapped by indirect end-labelling. Both treatments cause what appear to be strong double-stranded cleavages at 200 and 2200 base-pairs and at 17 X 10(3) base-pairs upstream from the rRNA transcription start. The cleavage at 200 base-pairs was analysed in greater detail using RNA hybridization probes specific for single DNA strands. The cleavage is in fact composed of three closely spaced nicks on each DNA strand. The DNA sequence at each of the nicks is strongly homologous across 15 base-pairs. Sodium dodecyl sulphate-induced cleavage by eukaryotic topoisomerase I is known to yield enzyme covalently attached to the 3' cut end of the DNA. We show that protein-linked DNA restriction fragments with their 3' ends at the cleavage sites are selectively retarded on denaturing gels, which provides strong evidence that the unusual cluster of cleavages is caused by a topoisomerase I. Additionally, the camptothecin results revealed cleavages not only at the specific upstream sites, but also across the transcribed region. Interestingly, the zone of camptothecin-assisted cleavage does not extend as far at the 3' end of the gene as the zone of endogenous nuclease sensitivity.
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PMID:Topoisomerase I cleavage sites identified and mapped in the chromatin of Dictyostelium ribosomal RNA genes. 283 75

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.
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PMID:Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro. 283 26

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.
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PMID:Topoisomerase II is a structural component of mitotic chromosome scaffolds. 298 25

Indirect end-labelling and the digestion patterns of endogenous and exogenous nucleases were used to analyse chromatin organization along the ribosomal RNA genes of Dictyostelium discoideum cells. A zone just upstream from the 5' end of the coding region was particularly sensitive to endogenous nucleases. In exponentially growing cells, this hypersensitive zone extended from -350 to -1600 bp relative to the transcription start. In sharp contrast, the DNA between 0 and -350 bp was strongly protected. In differentiating cells, in which the ribosomal RNA transcription rate is low, the 5' hypersensitive zone was more diffuse than in exponentially growing cells, and the protected region at the 5' end of the transcribed region was less pronounced. It is known that where DNA topoisomerase is acting on DNA, the addition of sodium dodecyl sulphate will result in cleavage of the DNA and covalent attachment of the enzyme to the cut DNA end. Treatment of nuclei from both exponentially growing cells and differentiating cells with SDS caused double-stranded cleavages at -200 (i.e. within the protected region), at -2200, and at two sites at about -17 kb. A fraction of the cleavage products appeared to be strongly associated with protein. Novobiocin, a DNA topoisomerase II inhibitor, did not inhibit the SDS-induced cleavages in vegetative cells. However, it significantly reduced the extent of nuclease cleavage within the -350 to -1600 bp hypersensitive zone. The possibility is discussed that there are two DNA topoisomerase-like activities on the ribosomal genes. One is site-specific and novobiocin-insensitive. We speculate that the other is responsible for maintaining DNA at the 5' end of the gene in a torsionally strained, nuclease-hypersensitive state.
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PMID:Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes. 301 83

We have examined DNA in cells treated with 5,6-dichloro-1-beta-O-ribofuranosylbenzimidazole (DRB), an adenosine analogue. The results show that DRB induces an partial fragmentation of DNA when the cells are lysed in dilute alkali. Fragmentation of DNA does not occur in control cells, nor in cells pretreated with novobiocin or VP-16/VM-26. The data show that DRB interferes with DNA topoisomerase II. In agreement with this interpretation, crude nuclear extracts of DRB-treated cells result in reduced in vitro KC1/SDS precipitation of covalent protein-DNA complexes. Formation of covalent complexes is typical of topoisomerase-DNA interaction.
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PMID:5,6-Dichloro-1-beta-O-ribofuranosylbenzimidazole induces DNA damage by interfering with DNA topoisomerase II. 303 22


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