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 mechanism by which the type 2 topoisomerase from bacteriophage T4 mediates knotting of negatively supercoiled DNA was deduced from an analysis of product topology. The knotted products were nicked and then subjected to electrophoresis in order to separate species on the basis of the minimum number of crossings in the knotted form. Knots with defined numbers of crossings were purified and the configuration of these crossings determined in the electron microscope by the RecA coating method. The product knots were exclusively of the twist form, in which an interwound region is entrapped by a single interlock of two looped ends. The interwound region was of negative sign in greater than 98% of the molecules examined, whereas the single interlock was equally likely to be positive or negative. These results are interpreted in terms of a model for knot formation in which random strand passage mediated by the topoisomerase links bent or branched portions of a superhelix that has a specific interwound geometry. Superhelix interwinding and DNA contacts stabilized by excess enzyme molecules explain the very high frequency of knotting.
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PMID:Supercoiled DNA-directed knotting by T4 topoisomerase. 165 29

Treatment of mammalian cells with DNA intercalating agents produces protein-associated DNA strand breaks. These breaks have been proposed to represent the action of a topoisomerase, which would alter the DNA linking number. Changes in DNA linking number in cells treated with the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) were studied by ethidium titration of nucleoid sedimentation. m-AMSA treatment was found to produce an increase in DNA linking number. Previously, we had proposed that intercalator-induced protein-associated DNA breaks act to reduce DNA torsional strain that results from the intercalator-induced decrease in DNA twist. In such a model, linking number would be expected to decrease. The finding that the DNA linking number increased following m-AMSA treatment suggests that intercalators may block enzymes that normally decrease linking number. Such enzymes would have DNA gyrase like properties. Consistent with this possibility, a DNA gyrase inhibitor, novobiocin, inhibited the restoration of normal linking number and, to a lesser degree, the reversal of protein-associated strand breaks after removal of intercalator.
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PMID:Changes in deoxyribonucleic acid linking number due to treatment of mammalian cells with the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide. 654 49

Streptonigrin stimulated unique intensity patterns of topoisomerase II-mediated DNA cleavage in agarose and sequencing gels with no similarity to those of doxorubicin, VM-26,4'(9-acridinylamino)-methanesulfon-m-anisidide, genistein, and mitoxantrone. Surprisingly, a statistical analysis of 60 sites stimulated by streptonigrin in SV40 and pBR322 DNAs showed that the drug required the dinucleotide 5'-TA-3' from 2- to 3-positions at the DNA cleavage site. Streptonigrin did not intercalate into the double helix; however, a positive value of the reduced linear dichroism indicated that indeed the drug interacted with the DNA. An angle of 45 degrees was found between the major drug and local DNA axes, suggesting a minor groove binding mode. Moreover, a DNA winding assay showed that streptonigrin may tighten the helical twist of DNA, similar to the known minor groove binder distamycin. Drug competition for receptor site binding was then evaluated by drug combination in the cleavage reaction. DNA cleavage intensity patterns were altered only with the streptonigrin/mitoxantrone combination, suggesting that the two compounds may compete for ternary complex formation. The results indicate that streptonigrin may bind to the DNA in a manner similar to that of minor groove binders and that its pharmacophore, possibly different from other topoisomerase II inhibitors, may be an important determinant of its unique sequence position specificity.
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PMID:Unique sequence specificity of topoisomerase II DNA cleavage stimulation and DNA binding mode of streptonigrin. 792 86

Bulgarein, a fungal metabolite, induced mammalian topoisomerase I-mediated DNA cleavage in vitro. The cleavage activity of bulgarein was comparable to that of camptothecin at a drug concentration range of 0.025-approximately 5 microM. The DNA cleavage induced by bulgarein was suppressed at concentrations above 12.5 microM. Treatment of a reaction mixture containing bulgarein and topoisomerase I with elevated temperature (65 degrees C) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I-mediated DNA cleavage induced by bulgarein is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex." Intensity of cleaved DNA fragments induced by bulgarein with topoisomerase I was different from that induced by camptothecin. Bulgarein inhibited catalytic activities of both topoisomerase I and topoisomerase II. The changes in absorption spectra of bulgarein in the visible region observed upon addition of increasing amounts of calf thymus DNA indicate that bulgarein interacts with DNA. DNA (un)winding assay by two-dimensional gel electrophoresis showed that bulgarein induced the winding of DNA in the opposite direction to that of an intercalator so that positively supercoiled molecules are produced. Thus, bulgarein represents a new class of drugs which stabilizes the cleavable complex of topoisomerase I and alters the structure of DNA in a manner leading to a tightening of the helical twist.
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PMID:Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein. 839 Apr 57

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins are in vitro substrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.
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PMID:Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2. 983 53

For a series of antitumor-active 5-substituted 9-aminoacridine-4-carboxamide topoisomerase II poisons, we have used X-ray crystallography and stopped-flow spectrophotometry to explore relationships between DNA binding kinetics, biological activity, and the structures of their DNA complexes. The structure of 5-F-9-amino-[N-(2-dimethylamino)ethyl]-acridine-4-carboxamide bound to d(CGTACG)(2) has been solved to a resolution of 1.55 A in space group P6(4). A drug molecule intercalates between each of the CpG dinucleotide steps, its protonated dimethylamino group partially occupying positions close to the N7 and O6 atoms of guanine G2 in the major groove. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Intercalation unwinds steps 1 and 2 by 12 degrees and 8 degrees, respectively compared with B-DNA, whereas the central TpA step is overwound by 10 degrees. Nonphenyl 5-substituents, on average, decrease mean DNA dissociation rates by a factor of three, regardless of their steric, hydrophobic, H-bonding, or electronic properties. Cytotoxicity is enhanced on average 4-fold and binding affinities rise by 3-fold, thus there is an apparent association between kinetics, affinity, and cytotoxicity. Taken together, the structural and kinetic studies imply that the main origin of this association is enhanced stacking interactions between the 5-substituent and cytosine in the CpG binding site. Ligand-dependent perturbations in base pair twist angles and their consequent effects on base pair-base pair stacking interactions may also contribute to the stability of the intercalated complex. 5-Phenyl substituents modify dissociation rates without affecting affinities, and variations in their biological activity are not correlated with DNA binding properties, which suggests that they interact directly with the topoisomerase protein.
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PMID:Acridinecarboxamide topoisomerase poisons: structural and kinetic studies of the DNA complexes of 5-substituted 9-amino-(N-(2-dimethylamino)ethyl)acridine-4-carboxamides. 1095 60

Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication. Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils. Here, we investigate the mechanism underlying this chiral preference. Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions. Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle.
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PMID:Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases. 1285 58

Escherichia coli topoisomerase IV (Topo IV) is an essential ATP-dependent enzyme that unlinks sister chromosomes during replication and efficiently removes positive but not negative supercoils. In this article, we investigate the binding properties of Topo IV onto DNA in the absence of ATP using a single molecule micromanipulation setup. We find that the enzyme binds cooperatively (Hill coefficient alpha approximately 4) with supercoiled DNA, suggesting that the Topo IV subunits assemble upon binding onto DNA. It interacts preferentially with (+) rather than (-) supercoiled DNA (Kd+=0.15 nM, Kd-=0.23 nM) and more than two orders-of-magnitude more weakly with relaxed DNA (Kd0 approximately 36 nM). Like gyrase but unlike the eukaryotic Topo II, Topo IV bends DNA with a radius 0= 6.4 nm and locally changes its twist and/or its writhe by 0.16 turn per bound complex. We estimate its free energy of binding and study the dynamics of interaction of Topo IV with DNA at the binding threshold. We find that the protein/DNA complex alternates between two states: a weakly bound state where it stays with probability p = 0.89 and a strongly bound state (with probability p = 0.11). The methodology introduced here to characterize the Topo IV/DNA complex is very general and could be used to study other DNA/protein complexes.
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PMID:Topoisomerase IV bends and overtwists DNA upon binding. 1586 84

Smc2/4 forms the core of the Saccharomyces cerevisiae condensin, which promotes metaphase chromosome compaction. To understand how condensin manipulates DNA, we used two in vitro assays to study the role of SMC (structural maintenance of chromosome) proteins and ATP in reconfiguring the path of DNA. The first assay evaluated the topology of knots formed in the presence of topoisomerase II. Unexpectedly, both wild-type Smc2/4 and an ATPase mutant promoted (+) chiral knotting of nicked plasmids, revealing that ATP hydrolysis and the non-SMC condensins are not required to compact DNA chirally. The second assay measured Smc2/4-dependent changes in linking number (Lk). Smc2/4 did not induce (+) supercoiling, but instead induced broadening of topoisomer distributions in a cooperative manner without altering Lk(0). To explain chiral knotting in substrates devoid of chiral supercoiling, we propose that Smc2/4 directs chiral DNA compaction by constraining the duplex to retrace its own path. In this highly cooperative process, both (+) and (-) loops are sequestered (about one per kb), leaving net writhe and twist unchanged while broadening Lk. We have developed a quantitative theory to account for these results. Additionally, we have shown at higher molar stoichiometries that Smc2/4 prevents relaxation by topoisomerase I and nick closure by DNA ligase, indicating that Smc2/4 can saturate DNA. By electron microscopy of Smc2/4-DNA complexes, we observed primarily two protein-laden bound species: long flexible filaments and uniform rings or "doughnuts." Close packing of Smc2/4 on DNA explains the substrate protection we observed. Our results support the hypothesis that SMC proteins bind multiple DNA duplexes.
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PMID:The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. 1610 Jan 11

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 microM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I-DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I-DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Delta39LS lacking 1-39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335-16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Delta39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I.
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PMID:Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. 1648 84


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