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)

The aim of our work was to investigate whether DNA topoisomerase II participates in the repair-specific incision of UV-irradiated genomic DNA. Therefore, the influence upon DNA incision of the topoisomerase II inhibitors (nalidixic and oxolinic acid, novobiocin and coumermycin A1) as well as the intercalating agent quinacrine has been measured in normal human fibroblasts using the alkaline elution technique. In addition, inhibition by novobiocin has been determined in fibroblast strains from 11 normal donors and from 16 xeroderma pigmentosum (XP) patients belonging to the complementation groups A, C, D, E, and XP variant. Nalidixic and oxolonic acid did not inhibit endonucleolytic cleavage, whereas novobiocin was a potent inhibitor of DNA incision. It was observed that in normal and in all XP strains 50% inhibition by novobiocin occurred on average in the dose range 315-590 microM. Since inhibition by novobiocin was not paralleled by that with the other topoisomerase II inhibitors nalidixic and oxolinic acid, it must be concluded that reduction of enzyme-catalysed breaks was not due to the participation of topoisomerase II in the incision step, but to the displacement of ATP at the binding site of the DNA-incising enzyme. This enzyme absolutely requires ATP as a cofactor for endonucleolytic cleavage. Quinacrine, however, inhibited DNA incision in normal fibroblasts at a mean Ki of 318 microM. Inhibition by this intercalating agent seems to be caused by structural perturbations in DNA, which render it a poor substrate for endonucleolytic cleavage.
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PMID:The effects of inhibitors of topoisomerase II and quinacrine on ultraviolet-light-induced DNA incision in normal and xeroderma pigmentosum fibroblasts. 184

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.
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PMID:Chloroplast DNA topoisomerase I from cauliflower. 184 83

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

DNA in bacterial cells is under negative superhelical tension, a feature that facilitates many of the activities of DNA. Supercoiling is introduced enzymatically by DNA gyrase, and the accumulation of excessively high levels is prevented by the relaxing activity of DNA topoisomerase I. Among the factors likely to influence supercoiling are topoisomerase gene expression, the ratio of ATP to ADP concentration, and processes such as transcription that unwind DNA and then translocate along it.
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PMID:Bacterial topoisomerases and the control of DNA supercoiling. 196 69

DNA topoisomerase-II activity was measured in a variety of rat organs and in two types of cultured mammalian cells at different stages of growth. The assay for enzyme activity is based on the ability of DNA topoisomerase II to catenate relaxed, circular double-stranded [3H]DNA into huge networks of interlocked circles which can be selectively trapped on a nitrocellulose filter. This catenation requires ATP and provides a sensitive, specific, and quantitative way to measure topoisomerase-II activity in crude extracts of nuclei. The level of type-II topoisomerase activity showed little variation at different stages of growth in either Chinese hamster ovary cells or human skin fibroblasts. In both cell types, growth-arrested cells contain levels of topoisomerase II very similar to those seen in actively growing cells. In addition, substantial levels of type-II topoisomerase are found not only in those rat organs expected to contain large populations of growing cells (testis, spleen), but also in organs composed primarily of cells in G0 (brain, liver, lung). These data indicate that total nuclear type-II topoisomerase activity does not vary dramatically with the state of cell growth or degree of cell differentiation.
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PMID:Identification of DNA topoisomerase-II activity in terminally differentiated mammalian organs and in non-growing cultured cells. 196 87

The cancer chemotherapeutic agent amsacrine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), is thought to effect cytotoxicity by inhibiting the ATP-dependent enzyme topoisomerase II in the act of its duplex strand-passing action. Upon protein denaturation, the arrested "cleavable complex" that results gives rise to double- and single-strand breaks (dsbs and ssbs) and DNA-protein cross-links (dpcs). Simultaneous cotreatments with 2,4-dinitrophenol (DNP) or novobiocin (novo) abrogates mAMSA cytotoxicity in Chinese hamster cells (H. Utsumi et al., Cancer Res., 50:2577-2581, 1990). Pulsed-field gel electrophoresis was used to estimate dsbs, velocity sedimentation in alkaline sucrose gradients for ssbs, and alkaline elution without protease digestion for dpcs. Although cotreatment with DNP or novo modulated somewhat the yield of DNA lesions due to mAMSA, quantitatively these changes did not correlate at all with, and therefore could not account for, the reduced lethality that resulted from cotreatments. For example, DNA cotreatment markedly increased the yields of dsbs, ssbs, and dpcs, even though cell killing was appreciably reduced. Furthermore, neither DNP nor novo cotreatment affected the rate, or the completeness of, the repair of mAMSA-induced DNA damage, and neither cotreatment lowered total cellular ATP. Hence, the arresting of the cleavable complex by mAMSA, made evident by lesions in DNA, did not correlate with cytotoxicity. However, cotreatment with either DNP or novo resulted in an enhanced recovery of the mAMSA-induced inhibition of replicative DNA synthesis. Because DNP and novo (transiently) slow down DNA synthesis, it is proposed that these compounds abrogate mAMSA killing of S phase cells by reducing the disorganization of the processing of replicated DNA by topoisomerase II.
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PMID:Amsacrine-induced lesions in DNA and their modulation by novobiocin and 2,4-dinitrophenol. 198 75

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
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PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52

Fostriecin causes a delayed inhibition of replicative DNA synthesis in human cells, consistent with a role for DNA topoisomerase II (its target enzyme) at a late stage in replication. Fostriecin does not inhibit UV-induced excision repair. The less specific inhibitor novobiocin blocks repair in permeabilised cells given a low dose of UV, presumably through a mechanism other than the inhibition of topoisomerase II. Its effect cannot be accounted for by a depletion of the ATP required for incision. Camptothecin, an inhibitor of DNA topoisomerase I, blocks replicative DNA synthesis immediately but incompletely, suggesting a participation of topoisomerase I at the replication fork, but it, too, has no influence on DNA repair. We thus find no evidence for involvement of either topoisomerase I or II in the response of cells to UV damage.
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PMID:Comparison of effects of fostriecin, novobiocin, and camptothecin, inhibitors of DNA topoisomerases, on DNA replication and repair in human cells. 215 21

Using cultured V79 Chinese hamster cells, we found that novobiocin (or 2,4-dinitrophenol) can abrogate, almost completely, the cytotoxicity due to the topoisomerase II inhibitor amsacrine (mAMSA). V79 cells were sensitive to mAMSA killing at all stages in the cell cycle but mainly in S phase followed by late G1 phase; however, novo rescued cells of all ages. The properties of two kinds of radiation-sensitive Chinese hamster cells were also examined, i.e., the line of V79 cells that can be rescued by caffeine, designated S-10 (H. Utsumi and M.M. Elkind, Radiat. Res., 96: 348-358, 1983); and Chinese hamster ovary cells (P.A. Jeggo and L.M. Kemp, Mutat. Res., 112: 313-327, 1983) which are also sensitive to other DNA-damaging agents. As is the case for exposure to radiation, after mAMSA treatment caffeine rescued V79/S-10 cells. Although Jeggo's Chinese hamster ovary cells were more responsive to mAMSA, novo still abrogated mAMSA toxicity in the mutant cells as well as in the parental Chinese hamster ovary cells 2,4-Dinitrophenol acted similarly to novo with respect to mAMSA killing, but neither compound reduced the ATP content of V79 cells. We propose that one reason for the rescue from mAMSA killing of at least S-phase cells by novo or 2,4-dinitrophenol is their ability transiently to inhibit replicative DNA synthesis.
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PMID:Abrogation by novobiocin of cytotoxicity due to the topoisomerase II inhibitor amsacrine in Chinese hamster cells. 215 94

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.
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PMID:Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies. 216 85


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