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)

Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme.
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PMID:The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA. 256 Jan 91

We showed previously that nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line, CCRF-CEM, exhibited decreased DNA topoisomerase II activity and ability to form drug-stabilized covalent protein-DNA complexes (Danks et al., Biochemistry 27:8861-8869; 1988). In the present study, we found that nuclear extracts of these sublines (approximately 50- and approximately 140-fold resistant to VM-26) required 2 and 8 times more adenosine 5'-triphosphate (ATP), respectively, to achieve half-maximal stimulation of unknotting activity compared to extracts from the sensitive cells. When novobiocin, a competitive inhibitor of ATP binding to topoisomerase II, was included in the reaction, this ATP requirement increased 2.5- to 4-fold with the CEM cell extracts and 3.5- to 12-fold with the resistant cell extracts. ATP produced a dose-dependent increase in VM-26-stabilized topoisomerase II-DNA covalent complexes with nuclear extracts from all three cell lines. Extracts from resistant cells, however, formed 40-80% fewer complexes than those from sensitive cells. A similar decrease was seen with 4'-[(9-acridinyl)amino]methanesulphon-m-anisidide, to which the cells are cross-resistant. With nuclear extracts from sensitive cells, the tetralithium salt of 5'-adenylylimidodiphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, was as effective as ATP in promoting the formation of drug-stabilized enzyme-DNA complexes. With extracts from the resistant cell nuclei, however, AMP-PNP was about half as effective as ATP in promoting complex formation. Our results demonstrate that the effect of ATP on strand passing activity of and drug-stabilized complex formation by topoisomerase II is decreased in the nuclear extracts from the drug-resistant cells and suggest a possible basis for this form of drug resistance.
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PMID:Increased ATP requirement for activity of and complex formation by DNA topoisomerase II from human leukemic CCRF-CEM cells selected for resistance to teniposide. 256 33

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.
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PMID:Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria. 282 74

Changes in the association of the catalytic subunit and the regulatory subunits of isozymes I and II of cAMP-dependent protein kinases (RI and RII, respectively) with the transcriptionally active chromatin fraction from rat liver were examined after a glucagon/theophylline injection and also after partial hepatectomy. Chromatin was partitioned into transcriptionally active and bulk, transcriptionally inactive fractions by digestion with micrococcal nuclease under appropriate conditions. In both experimental models, an increased content of catalytic and both RI and RII subunits was observed in chromatin fractions that were enriched in transcriptionally active DNA, particularly in the fraction associated with the residual nuclear matrix-lamina. The changes in the association of the subunits with these fractions paralleled the increases in intracellular cAMP levels and occurred in a time frame compatible with the changes in gene expression. The catalytic subunits could be removed from the nuclear matrix-lamina fraction by salt, whereas the two regulatory subunits remained tightly bound. The data support the concept of a direct role of the regulatory subunits of cAMP-dependent protein kinases in the induction of gene expression. However, we were unable to confirm that RII possessed an intrinsic topoisomerase activity.
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PMID:The regulatory and catalytic subunits of cAMP-dependent protein kinases are associated with transcriptionally active chromatin during changes in gene expression. 289 95

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.
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PMID:Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation). 299 83

The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription.
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PMID:Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus. 300 75

Aza-ellipticines are DNA intercalative ellipticine analogues with antitumor activity that induce protein-linked DNA breaks in NIH 3T3 cells in culture. The effects of two aza-ellipticine congeners (BD-40 and BR-76) on the activity of purified Calf Thymus type II topoisomerase were studied using pUC13 DNA as substrate. DNA cleavage was stimulated by both molecules at those doses required for inducing lethal effects in cells (DE5O). This effect was reversed by high salt treatment, indicating that it was actually mediated by Topo II. Mapping of cleavage sites on linearized and 3' end-labelled pUC13 DNA showed that ellipticine and aza-ellipticines stimulated the same sites, which differed from those stimulated by m-AMSA. Decatenating activity of Topo II on Trypanosoma cruzi kDNA was both inhibited by ellipticine and BD-40 at concentrations much higher than DE50 concentrations. Activity of aza-ellipticines was also investigated on isolated nuclei. Unlike ellipticine which promoted DNA-breaking activity, BD-40 and BR-76 were repeatedly inactive. Prior treatment of DNA by Proteinase K did not reveal hidden breaks which are formed in intact cells treated with BD-40 (Vilarem et al., 1984, Nucleic Ac. Res. 12, 8653). Concordant with these data, BD-40 did not impair DNA-synthetic activity in isolated nuclei, while Ellipticine largely decreased it. These results indicate that lesions induced in DNA by Aza-ellipticines are mediated by Topo II. The absence of effect of these drugs on isolated nuclei compared to that of Ellipticine may be due to some specific features of the association between Topo II and Aza-ellipticines or reflect a bioactivation step as a prerequisite for in vivo activity.
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PMID:The in vitro involvement of topoisomerase II in the activity of aza-ellipticine analogues is not correlated with drug activity on isolated nuclei. 301 46

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.
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PMID:Eukaryotic topoisomerase II. Characterization of enzyme turnover. 301 13

DNA topoisomerase II was purified from calf thymus nuclei by a simple and fast four-step procedure: selective ammonium sulfate precipitation, chromatography on blue-Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on ATP or dATP, the Km values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5-8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaCl. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.
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PMID:Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa. 302 77

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.
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PMID:Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin. 354 Sep 37


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