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)

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Linking number anomalies in DNA under conditions close to condensation. 265 31

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases alpha and delta, topoisomerase II, and replication protein A, (RP-A).
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PMID:Cofractionation of HeLa cell replication proteins with ors-binding activity. 767 29

Two synthetic DNA molecules that can be knotted have been employed as substrates for E. coli DNA topoisomerases I and III. Both molecules contain 104 nucleotides, including sequences that can form two single-turn helical domains, connected by single-stranded oligo(dT) linkers in an X-Y-X'-Y' pairing motif. One of the knots can be ligated to form cyclic molecules with the topologies of a circle, a trefoil knot with negative nodes, or a figure-8 knot. Cyclic molecules constructed from the other molecule can form a circle, a figure-8 knot, and trefoil knots with either positive or negative nodes. The topologically negative nodes in these knots are derived from right-handed B-DNA, and the positive nodes are derived from left-handed Z-DNA. The topoisomerases can catalyze the interconversion of the different topological forms of these molecules, as a function of solution conditions and the extent to which they favor B-DNA or Z-DNA. The enzymes appear to catalyze a single strand-passage event at a time. The topoisomerases can catalyze strand passage events involving both positive and negative nodes as substrates. Gel retention experiments show that both knots can bind up to four molecules of E. coli DNA topoisomerase I. The thermal denaturation of the domains of a trefoil knot closely related to these knots suggests that the two helical domains are uncoupled, so the single-stranded linkers in the knots are not taut. Chemical ligation experiments yield a distribution of products similar to those of enzymatic ligation, showing that the ATP cofactor in DNA knot ligation does not appear to skew the products markedly. Knots that are stressed by being placed in unfavorable solution conditions have been shown to be a highly sensitive system for detecting topoisomerase activity.
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PMID:Topological transformations of synthetic DNA knots. 781 63

Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts. 792 26

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.
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PMID:Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain. 849 Jan 85

In the MCF-7 human breast [correction of beast] adenocarcinoma cell line, acute exposure to 1 muM doxorubicin inhibited cell proliferation by approximately 75%. Analysis of cell cycle distribution indicated that within 24 hr, the G(2)/M fraction increased more than 3-fold and the S-phase population declined by >50%. In addition to growth arrest, there was an approximately 40% reduction in the viable cell population after 72 hr. Gel electrophoretic resolution of low molecular weight DNA immediately after exposure of cells to doxorubicin failed to demonstrate "laddered" oligonucleosomal profiles associated with apoptosis. The absence of intracellular DNA fragments or release of fragmented DNA into the incubation medium was confirmed by spectrofluorophotometry over a 72 hr interval following exposure of cells to 1 muM doxorubicin. In addition, there was no evidence of the morphological features associated with apoptosis during this period. Acute exposure to 1 muM doxorubicin also produced a transient increase in c-myc message expression (within the first hour) followed by a decline to 70% of control levels within 2-4 hr. The reduction in c-myc mRNA levels was concentration dependent and corresponded closely with growth arrest (as well as with inhibition of DNA synthesis). These findings (as well as similar reports demonstrating a correspondence between reduced c-myc expression and growth inhibition by VM-26 and m-AMSA in MCF-7 cells) suggest that the down-regulation of c-myc expression may reflect perturbations in regulatory processes contributing to growth arrest in MCF-7 cells exposed to topoisomerase II inhibitors.
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PMID:Growth arrest and non-apoptotic cell death associated with the suppression of c-myc expression in MCF-7 breast tumor cells following acute exposure to doxorubicin. 865 43

Linearized free maxicircle DNA, present in detergent lysates of Crithidia fasciculata mitochondria, was thought to be a replication intermediate formed during rolling circle replication of maxicircle DNA. Gel electrophoresis of the linearized free maxicircles indicated that they were slightly larger than the maxicircle genome, raising the possibility of the presence of terminal repetitions (Hajduk, S.L., Klein, V.A. and Englund, P.T. (1984) Cell 36, 483-492). We recently found, however, that maxicircles replicate by a theta-mechanism, and not as rolling circles (Carpenter, L.R. and Englund, P.T. (1995) Mol. Cell Biol. 15, 6794-6803). Given that theta-replication does not easily explain the presence of linearized free maxicircles, we investigated alternative explanations for their existence. We present evidence that this DNA species results from the double-strand cleavage of maxicircles due to detergent denaturation of intracellular topoisomerase II cleavable complexes. As expected for a topoisomerase II cleavage product, the linearized free maxicircle DNA is covalently bound to protein at both 5' ends. In addition, the slightly larger apparent size of linearized free maxicircle DNA or maxicircles linearized by a restriction enzyme can be explained by anomalous electrophoretic migration during conventional or pulsed-field agarose gel electrophoresis. This anomalous migration is presumably due to bends or other unusual structures in the DNA.
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PMID:Linearized free maxicircle DNA in Crithidia fasciculata is a product of topoisomerase II-mediated cleavage. 892

The mechanism of action of a group of anthracene-containing analogs of amonafide was studied in Chinese hamster ovary (CHO) cells. These agents differ structurally from amonafide by the replacement of the naphthalene chromophore with an anthracene chromophore, the lack of a primary amine moiety in the 5 position, and substitutions at the 6 and 7 positions on the anthracene nucleus. In this study, five analogs with potent growth inhibitory activity and with low cardiotoxicity were chosen. Cytotoxicity analyses with tetrazolium dye assays (MTT) in vitro and continuous drug exposure revealed IC50 values in CHO cells in the nanomolar range. Intracellular scanning laser confocal microscopy of these drug-treated CHO cells showed that all analogs are able to enter cell nuclei with varying nuclear/cytoplasmic distribution: the more potent dimethylaminoethyl substituted analogs, 47 and 104, were primarily localized in the nucleus. Three analogs, including the unsubstituted parent (1), and numbers 35 (6-amino substituted) and 53 (6-aminoethyl substituted) inhibited DNA and RNA synthesis when assayed immediately after a 1 h exposure. In contrast, analogs 47 and 104 required 24 h post-drug exposure for 1 h to inhibit DNA and RNA synthesis. Using alkaline elution techniques, each analog also produced DNA single- and double-stranded breaks, as well as DNA protein cross-links. Interestingly, the most cytotoxic analogs, 47 and 104, produced minimal DNA strand damage in CHO cells at their IC90 concentrations, whereas the three other compounds with lower growth inhibitory potency produced marked and roughly equivalent DNA damage at equitoxic concentrations. Gel shift analysis of SV40 DNA exposed to the compounds demonstrated that these agents do not directly induce DNA strand breaks. However, catalytic studies with purified human topoisomerase II (Topo II) and plasmid DNA demonstrated that these drugs inhibit this enzyme. These results suggest that the azonafides inhibit Topo II to cause protein-associated strand breaks and impaired DNA and RNA synthesis. However, other mechanisms may also be operant, especially with the more potent dimethylamino ethyl substituted analogs.
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PMID:In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2'-(dimethylamino)ethyl]-1, 2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones with substitutions at the 6 and 7 positions (azonafides). 909 29

Mammalian cells express two isoforms of type II DNA topoisomerase which are the intracellular targets of many structurally diverse antineoplastic agents. The levels of topoisomerase II isozymes are important determinants for the sensitivity of cells to the cytotoxicity of drugs that target topoisomerase II. To investigate whether the expression of topoisomerase II isoforms is coordinated and the mechanisms governing their expression in the context of drug resistance, the 5'-flanking sequence for the gene of human topoisomerase IIbeta isoform was cloned and characterized. The 5'-flanking region has a very high GC content and contains no canonical TATA box element. Two separate transcriptional start sites are located to an adenine and a guanine, 193 and 89 nucleotides, respectively, upstream from the ATG translation initiation codon. Except for a small region immediately upstream of the translation initiation codon, there is no obvious sequence homology between the 5'-flanking sequences of human topoisomerase IIbeta gene and the previously described alpha gene. Transient expression assays with different 5'- and 3'-deletions of the 5'-flanking region of the topoisomerase IIbeta gene have delineated regions important for transcriptional regulation of the gene. Interestingly, sequences within the first intron also contribute to the promoter activity. Gel mobility shift studies demonstrate that protein factors from the nuclear extracts can bind specifically to the downstream elements and may participate in transcriptional regulation.
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PMID:Cloning and characterization of the 5'-flanking sequence for the human DNA topoisomerase II beta gene. 942 41

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.
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PMID:Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action. 1114 91


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