Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA untwisting enzyme has been purified approximately 300-fold from rat liver nuclei. The protein is greater than 90% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis. The native enzyme has a molecular weight between 64 000 and 68 000 and is composed of a single polypeptide chain. Evidence is presented that the protein can act catalytically. A trace amount of endonuclease activity associated with the most pure fraction could be a contaminant or it could be due to the action of the DNA untwisting enzyme itself.
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PMID:Purification and characterization of the DNA untwisting enzyme from rat liver. 18 21

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.
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PMID:DNA-relaxing enzyme from Micrococcus luteus. 20 27

Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.
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PMID:T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity. 22 76

In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.
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PMID:Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases. 131 8

Candida albicans is an opportunistic pathogen responsible for life-threatening infections in persons with impaired immune systems. Topoisomerase I is a potential target for novel antifungal agents; however, in order for this enzyme to be a therapeutically useful target, it needs to be demonstrated that the fungal and human topoisomerases differ sufficiently as to allow the fungal topoisomerase to be selectively targeted. To address this question, we isolated the topoisomerase I from C. albicans and compared its biochemical properties with those of the mammalian enzyme. Similar to other eukaryotic type I topoisomerases, the C. albicans type I topoisomerase has an apparent molecular mass of 102 kDa and covalently links to the 3' end of DNA, as shown after the reaction is interrupted by sodium dodecyl sulfate. Topoisomerase poisons such as camptothecin act by stabilizing the cleavage complex formed by the topoisomerase I and DNA. We observed that the C. albicans and mammalian type I topoisomerases differ in that the C. albicans cleavage complex is approximately 10-fold less sensitive to camptothecin than the mammalian cleavage complex is. In addition, we found that the antifungal agent eupolauridine can stabilize the cleavage complex formed by both the C. albicans and human topoisomerases and that the response of the C. albicans topoisomerase I to this drug is greater than that of the human enzyme. Thus, the topoisomerase I from C. albicans is sufficiently distinct from the human enzyme as to allow differential chemical targeting and will therefore make a good target for antifungal drug discovery.
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PMID:Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery. 133 88

A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.
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PMID:Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. 135 31

The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals. These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction. The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells. They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K(+)-ATPase loci or to cause chromosome aberrations. It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain antitumour compounds. The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials. The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target. Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds.
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PMID:Mutagenicity of quinolone antibacterials. 150 68

The object of this study was to devise a purification method for DNA/topoisomerase II complexes, with which to examine the enzyme's cleavage site specificity in cellular differentiation. Retinoic acid-induced differentiation involves topoisomerase II-mediated transient changes in DNA supercoiling, but it is not known whether this occurs at specific sites in the genome. Topoisomerase II forms a covalent DNA enzyme complex as it acts, which can be recovered by the sodium dodecyl sulfate (SDS)/KCl precipitation method, but this method fails to recover significantly more DNA from cells induced to differentiate. This may in part reflect the low numbers of retinoic acid-induced protein-linked breaks in DNA and also the method's relative inefficiency for DNA with few attached topoisomerase molecules. This suggested that an additional purification method would be required to enrich sufficiently for cleavage site DNA to address the issue of site specificity. The principle of our method is to couple poly(ethylene glycol) (PEG) to topoisomerase while it is covalently attached to DNA and then to use phase partitioning in an aqueous two-phase system of PEG and phosphate to separate free DNA from DNA bound to PEG-modified topoisomerases (which have high affinities for the phosphate-rich and PEG-rich phases, respectively). The method can be used in conjunction with DNase protection and, unlike the SDS/KCl method, can fractionate short fragments of DNA to which single protein molecules are attached. Using the SDS/KCl precipitation and new method in series, we have recovered protein-linked DNA from HL60 cells induced to differentiate to the granulocyte lineage (by retinoic acid) or to the monocyte/macrophage lineage (by phorbol myristate acetate) and have demonstrated that specific sequences become protein linked, probably to topoisomerase II, during induced differentiation.
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PMID:A method for the purification of DNA/protein complexes applied to DNA topoisomerase II cleavage sites. 164 31

To study the mechanism of illegitimate recombination in mammalian cells, we have developed a shuttle vector, pNK1, that contains three bacterial markers, amp (ApR), galK, and neo (KmR). The frequency of deletions occurring in autonomously replicating pNK1 DNA during the growth of monkey COS1 cells was measured by transfecting the plasmid into Escherichia coli cells and counting the number of galK- ApS double mutants among total KmR cells. This method allowed us to test the effects of topoisomerase inhibitors on deletion formation in mammalian cells. The DNA topoisomerase II (TopII) inhibitor, 4'-dimethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM26), stimulated deletions in pNK1 DNA in monkey cells. Since VM26 does not inhibit the strand-break activity of TopII, but rather stabilizes an enzyme-DNA complex in which DNA is cleaved upon treatment with sodium dodecyl sulfate, it is implicated that TopII participates in the deletion process in mammalian cells.
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PMID:A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency. 164 63

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.
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PMID:Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo. 165 19


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