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 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

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.
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PMID:DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA. 21 32

A guanine-rich single-stranded DNA from the human immunoglobulin switch region was shown by Sen and Gilbert [Nature, (1988) 334, 364-366] to be able to self-associate to form a stable four-stranded parallel DNA structure. Topoisomerase II did not cleave the single-stranded DNA molecule. Surprisingly, the enzyme did cleave the same DNA sequence when it was annealed into the four-stranded structure. The two cleavage sites observed were the same as those found when this DNA molecule was paired with a complementary molecule to create a normal B-DNA duplex. These cleavages were shown to be protein-linked and reversible by the addition of salt, suggesting a normal topoisomerase II reaction mechanism. In addition, an eight-stranded DNA molecule created by the association of a complementary oligonucleotide with the four-stranded structure was also cleaved by topoisomerase II despite being resistant to restriction endonuclease digestion. These results suggest that a single strand of DNA may possess the sequence information to direct topoisomerase II to a binding site, but the site must be base paired in a proper manner to do so. This demonstration of the ability of a four-stranded DNA molecule to be a substrate for an enzyme further suggests that these DNA structures may be present in cells.
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PMID:Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes. 131 62

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Features of apoptotic cells measured by flow cytometry. 133 43

Resistance to 0.8 microM 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) was induced by stepwise increases of drug concentration in the human tumor cell line CALc18 originating from a breast adenocarcinoma. The resistant cell line CALc18/AMSA exhibited a resistance index of 10 and a cross-resistance to other topoisomerase II inhibitors. A 3-fold decrease in the levels of topoisomerase II decatenating activity was found in CALc18/AMSA cells. By contrast, topoisomerase I activity was increased by about 3-fold in resistant cells. Interestingly this line was hypersensitive to camptothecin, a specific inhibitor of topoisomerase I. Restriction endonuclease patterns of the topoisomerase I and topoisomerase II loci were found to be identical in CALc18/AMSA and CALc18 with no evidence of gene amplification and rearrangements. Alkaline elution of m-AMSA-treated cells showed that DNA single strand breaks and DNA-protein crosslinks were decreased in CALc18/AMSA. The DNA lesions also obtained in m-AMSA-treated nuclei indicated that no drug uptake modification occurred in both cells. Moreover, the in vitro m-AMSA-induced DNA cleavage per unit of decatenating activity and the inhibitory effects of antitumoral drugs on decatenation were not found to be different with topoisomerase II from sensitive or resistant cells. However the specific cleavage induced by m-AMSA/per mg of crude protein from resistant cells was 2 to 3 times decreased. Multidrug resistance gene transcripts were not detected while levels of acidic glutathione S transferase mRNA were found to be 8 to 10-fold greater in resistant than in sensitive cell line with no amplification of the gene. In conclusion, the diminution of topoisomerase II activity and the increase of both topoisomerase I and acidic glutathione S transferase transcripts could contribute to the resistant phenotype of these breast cancer cells.
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PMID:Study of molecular markers of resistance to m-AMSA in a human breast cancer cell line. Decrease of topoisomerase II and increase of both topoisomerase I and acidic glutathione S transferase. 164 55

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.
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PMID:Potassium permanganate as an in situ probe for B-Z and Z-Z junctions. 166 68

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.
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PMID:Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication. 182 59

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.
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PMID:Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. 184 55

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

In previous studies we used Southern blotting to examine the topoisomerase II locus (on chromosome 17) in human leukemia cell lines and noted a difference in the XmnI restriction endonuclease digestion pattern between an m-AMSA-resistant line and its m-AMSA-sensitive parent line (Zwelling, L. A.; Hinds, M,; Chan, D.; Mayes, J.; Sie, K. L.; Parker, E.; Silberman, L.; Radcliffe, A.; Beran, M.; Blick, M. Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II. Journal of Biological Chemistry 264:16411-16420; 1989). We now demonstrate that the variable XmnI digestion pattern represents a normal restriction fragment length polymorphism (RFLP) which is observed in subjects without malignant disease and exhibits an autosomal pattern of inheritance. These data suggest that the previously described deviation in the genomic structure of topoisomerase II in the m-AMSA-resistant cell line did not reflect a new mutation, but rather a reduction to homozygosity at the topoisomerase II locus. This reduction to homozygosity is not due to chromosomal loss, as chromosome 17-specific gene probes clearly identify two chromosome 17's in the sensitive line and four in the resistant line, using chromosome painting with a chromosome 17-specific library. Some other genetic change must be the cause of the resistance of HL-60/AMSA and its topoisomerase II to the inhibiting actions of m-AMSA.
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PMID:A restriction fragment length polymorphism for human topoisomerase II: possible relationship to drug-resistance. 197 87


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