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 impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.
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PMID:Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16. 970 20

Psorospermin, a plant-derived antitumor agent, has been shown to selectively alkylate a guanine at the topoisomerase II cleavage site to trap the topoisomerase II-DNA cleaved complex. The results of this study provide further important insight into the mechanism of the topoisomerase II site-directed alkylation of DNA by psorospermin and its subsequent effects on the topoisomerase II-induced DNA cleavage. First, we demonstrate that the topoisomerase II-induced alkylation of DNA by psorospermin occurs at a time preceding the topoisomerase II-mediated strand cleavage event, because it occurs in the absence of Mg2+. We confirm that the alkylation of DNA by psorospermin takes place at N-7 of guanine in the presence of topoisomerase II, because substitution of the target guanine by 7-deazaguanine prevents alkylation. Because the stimulation of the topoisomerase II-induced DNA cleavage by psorospermin can be slowly reversed by the addition of excess salt, this indicates that alkylation of DNA by psorospermin traps a reversible topoisomerase II-DNA complex. Both the DNA alkylation by psorospermin in the presence of topoisomerase II and the enzyme-mediated DNA cleavage elevated by psorospermin are more enhanced at acidic pH values, in accordance with the increased stability of the topoisomerase II-DNA complex at acidic pH values. Finally, our results suggest that it is the psorospermin-DNA adducts, not the abasic sites resulting from depurination, that are responsible for the stimulation of the topoisomerase II-mediated cleavage. Because the precise location of the psorospermin within the topoisomerase II cleavage site is known, together with the covalent DNA linkage chemistry and the conformation of the psorospermin-DNA adduct, this structural insight provides an excellent opportunity for the design and synthesis of new, more effective topoisomerase II poisons.
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PMID:Topoisomerase II site-directed alkylation of DNA by psorospermin and its effect on topoisomerase II-mediated DNA cleavage. 983 55

Used for centuries in traditional Chinese medicine, camptothecin was rediscovered in the 1950s during a search for compounds that could be used as a source for steroid synthesis. Due to its limited water solubility, a sodium salt was used in the early clinical trials. The severe toxicity and erratic absorption relegated this compound to the research laboratory until the 1980s when the topoisomerase enzyme was identified as the cellular target of camptothecin, the topoisomerase enzyme was found to be overexpressed in cancer cells and a structure-activity relationship was determined for camptothecin. These new developments brought the camptothecins back to the clinical setting for further testing. The various analogues that have been most studied to date include: irinotecan (CPT-11), and its derivative SN-38, topotecan, and 9-aminocamptothecin. Numerous trials have been conducted in an attempt to establish the efficacy in various tumour types, to determine the dose-limiting toxicity and to define the optimal schedule of administration. It seems that large doses of these drugs given on intermittent schedules are not effective. Our hypothesis is that the camptothecins require a prolonged schedule of administration given continuously at low doses or frequent intermittent dosing schedules to be most effective. With these schedules, normal haematopoietic cells and mucosal progenitor cells with low topoisomerase I levels may be spared, while efficacy is preserved.
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PMID:Camptothecins: a review of their development and schedules of administration. 989 20

A 30-kDa DNA topoisomerase has been purified to near homogeneity from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. The enzyme is recognized by an antibody against a 16-mer peptide sequence from human DNA topoisomerase I. The purified enzyme is a type I topoisomerase. Consistent with the properties of other prokaryotic type I DNA topoisomerases, the isolated enzyme is unable to relax positively supercoiled DNA and absolutely requires divalent cations for its relaxation activity. However, regardless of the Mg+2 concentrations, ATP concentrations above 5 mM completely inhibit the relaxing activity. The enzyme is sensitive to high salt concentrations and the optimal activity occurs at salt concentrations between 3 and 30 mM for monovalent cations. Single-stranded M13 DNA is a strong inhibitor of this relaxing activity. The enzyme is inhibited by ethidium bromide, confirming that this DNA topoisomerase is incapable of relaxing positive supercoils. Topoisomerase I-specific inhibitors like Hoechst 32258 and actinomycin D inhibit the enzymatic activity while the enzyme is resistant to type II topoisomerase inhibitors such as norfloxacin, nalidixic acid, and novobiocin. From these enzymatic characteristics, we conclude that the R. capsulatus DNA topoisomerase is a prokaryotic type I DNA topoisomerase.
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PMID:Rhodobacter capsulatus DNA topoisomerase I purification and characterization. 991 36

We have used gel retardation analysis to show that human DNA topoisomerase IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase IIbeta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIbeta binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase IIbeta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIbeta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIbeta was able to cleave three of these four predicted sites. This indicates that topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase IIbeta with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.
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PMID:Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro. 992 30

A number of indenoisoquinolines were prepared and evaluated for cytotoxicity in human cancer cell cultures and for activity vs topoisomerase 1 (top1). The two most cytotoxic indenoisoquinolines proved to be cis-6-ethyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (21) and cis-6-allyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22), both of which displayed submicromolar mean graph midpoints when tested in 55 human cancer cell cultures. Two of the most potent top1 inhibitors were 6-(3-carboxy-1-propyl)-5,6-dihydro-5, 11-dioxo-11H-indeno[1,2-c]isoquinoline (26) and 6-ethyl-2, 3-dimethoxy-8,9-(methylenedioxy)-11H-indeno[1,2-c]isoquinolinium chloride (27), both of which also inhibited top2, unwound DNA, and are assumed to be DNA intercalators. However, two additional potent top1 inhibitors, 6-allyl-5,6-dihydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13c) and 5,6-dihydro-6-(4-hydroxybut-1-yl)-2,3-dimethoxy-8, 9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (19a), did not unwind DNA and did not affect top2. Some of the DNA cleavage sites detected in the presence of the indenoisoquinolines were different from those seen with the camptothecins. The cleavage sites induced by the indenoisoquinolines were reversed by salt treatment, which is consistent with the reversible trapping of top1 cleavable complexes by the indenoisoquinolines. In general, the potencies of the indenoisoquinolines as top1 inhibitors did not correlate with their potencies as cytotoxic agents, as some of the most cytotoxic agents had little if any effect on top1. On the other hand, the most potent of the indenoisoquinolines vs top1 were not the most cytotoxic. In several cases, moderate activity was observed for both cytotoxicity and activity vs top1.
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PMID:Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons. 998 16

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.
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PMID:Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix. 1032 34

Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation.
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PMID:Nucleosome dynamics. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle. 1045 91

DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme.
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PMID:The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps. 1049 25

The Bloom (BLM) and Werner's (WRN) syndrome proteins may regulate recombination and DNA repair. Using a novel polyclonal antibody to human BLM, we detected the 170-kda BLM antigen in wild-type but not Bloom syndrome cells. BLM was localized to punctate nuclear structures. The level of BLM but not WRN was 3.6 fold-higher in G(1)/S-synchronized fibroblasts than in G(0)-synchronized fibroblasts. BLM-positive cells invariably expressed topoisomerase IIalpha, whereas topoisomerase IIbeta was expressed constitutively. Transfections of BLM deletion mutants demonstrated that the C-terminal domain of BLM mediated nuclear entry and the central helicase domain was necessary for producing the punctate pattern. By subcellular fractionation, BLM was found primarily in high-salt extracts of the nucleoplasm and the nuclear matrix and was enriched in G(1)/S-synchronized cells compared with G(0)-synchronized cells. There was no interaction between BLM and WRN or topoisomerases IIalpha and IIbeta in fibroblasts. These results demonstrate that BLM is targeted to specific nuclear structures and that its expression is enhanced during cell growth. The known nucleolar localization of WRN, its invariant expression during the cell cycle, and the lack of interaction between BLM and WRN suggest distinct roles for BLM and WRN in processes such as DNA repair and recombination.
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PMID:Localization of the Bloom syndrome helicase to punctate nuclear structures and the nuclear matrix and regulation during the cell cycle: comparison with the Werner's syndrome helicase. 1056 3


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