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)

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

We have previously shown that a cloned 480 bp DNA fragment that spans the 3'-enhancer region of the avian beta-globin gene cluster can become very tightly, perhaps covalently, bound to protein in avian nuclear matrices in vitro [Zenk et al. (1990) Biochemistry 29, 5221-5226]. This binding was not tissue-specific and was probably not mediated by topoisomerase enzymes. In the present study, we have examined avian nuclear matrices (or scaffolds) for the presence of very tight cellular DNA-protein complexes in the region of the beta-globin gene enhancer and of several other avian genes. Nuclear matrices were prepared by both high- and low-salt methods, and protein-DNA complexes were isolated by SDS/K+ precipitation after restriction enzyme digestion. In adult reticulocytes, up to 30% of the intact 3800 bp HindIII-EcoRI fragment that encompasses the beta-globin enhancer element may be very tightly bound to nuclear matrix protein. In adult avian thymus nuclei, the beta-globin enhancer is neither matrix-associated nor tightly bound to protein. In contrast, a 5.0-kb HindIII fragment of the malic enzyme gene is very tightly bound to nuclear matrix-associated protein in thymus cells, but not reticulocytes. The malic enzyme gene is active in thymus cells, and not in reticulocytes. These results suggests that certain regions of cellular DNA are very tightly, perhaps covalently, attached to nuclear matrix-associated proteins. Attachment follows a tissue-specific pattern that is associated with transcriptional activity.
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PMID:Avian nuclear matrix proteins bind very tightly to cellular DNA of the beta-globin gene enhancer in a tissue-specific fashion. 204 26

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

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

We obtained a clinical isolate of Providencia stuartii showing a high level of resistance to norfloxacin and to other 4-quinolones, whose target is the enzyme DNA-gyrase. This strain showed resistance also to beta-lactam and aminoglycoside antibiotics. In order to detect modification of DNA-gyrase, we performed supercoiling assays in vitro in presence of norfloxacin and ciprofloxacin. Furthermore, outer membrane proteins, which are involved in permeability mechanisms, were analyzed on SDS-polyacrylamide gels. Results showed that both modifications in DNA-gyrase and changes in outer membrane proteins can be held responsible for resistance to 4-quinolones; moreover, these modification are probably supported by a third mechanism of resistance.
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PMID:Modifications of DNA-gyrase and of permeability in a norfloxacin-resistant clinical isolate of Providencia stuartii. 282 73

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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