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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.
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PMID:A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II. 165 25

Podophyllotoxin (PD) and its derivative etoposide (VP-16), a clinically useful anticancer drug, exhibit different mechanisms of action. PD binds specifically to tubulin to prevent its polymerization, whereas VP-16 lacks this action. The DNA strand breakage caused by VP-16 is thought to be due to its interaction with topoisomerase II or to free radical formation by oxidation of its 4'-phenolic hydroxyl group to a semiquinone free radical. We have demonstrated that PD, VP-16, 4'-demethylepipodophyllotoxin (DEPD), and syringic acid (SA) exhibit no DNA-cleaving activity but, in the presence of metal ions such as Cu2+ and Fe3+, DEPD and SA form metal complexes, which in turn show high activity for DNA strand scission at pH 7.8 under air. Furthermore, it was found that DNA cleavage was greatly promoted by irradiation with UV light. The PD-Fe3+ system at pH 7.8 showed very low DNA-cleaving activity, but irradiation with UV light in the system induced almost complete DNA breakage. DNA cleavages were significantly inhibited in the presence of hydroxyl radical scavengers, such as sodium benzoate and dimethylurea, in the Cu(2+)-SA and Fe(3+)-PD systems, with or without UV irradiation. These reactions were investigated by optical and ESR spectra, coupled with ESR spin-trapping techniques, by which the formation of hydroxy radicals was clearly detected in all systems. These findings have led us to a new proposal of the metal- and photo-induced mechanism for understanding the antitumor action of PD, VP-16, and their related compounds.
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PMID:Metal- and photo-induced cleavage of DNA by podophyllotoxin, etoposide, and their related compounds. 175 45


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