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)

A novel ATP-dependent DNA topoisomerase which makes reversible double-strand breaks in the DNA double helix has been purified to near homogeneity from T4 bacteriophage-infected Escherichia coli cells. Genetic data suggest that this activity is essential for initiating T4 DNA replication forks in vivo.
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PMID:T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication. 22 89

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

The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA.
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PMID:Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca). 131 54

Four naturally occurring flavones (baicalein, quercetin, quercetagetin and myricetin) and two novel catechins [(-)-epicatechin gallate and (-)-epigallocatechin gallate, from the tea plant Camellia sinensis], which are known inhibitors of reverse transcriptase, were shown to induce mammalian topoisomerase II-dependent DNA-cleavage in vitro. The flavones differed from the catechins in causing unwinding of duplex DNA, but both classes of compound induced enzymic DNA breakage at the same sites on DNA. Moreover, the cleavage specificity was the same as that for the known intercalator 4'-(acridin-9-ylamino)methanesulphon-m-anisidide, suggesting that these agents trap the same cleavable complex. Analysis of some 30 flavonoid compounds allowed elucidation of the structure-function relationships for topoisomerase II-mediated DNA cleavage. For flavonoid inhibitors an unsaturated double bond between positions 2 and 3 of the pyrone ring and hydroxy groups at the 5, 7, 3' and 4' positions favoured efficient cleavage. Hydroxy substitutions could be tolerated at the 3, 6 and 5' positions. Indeed, the absence of substituents at the 3', 4' and 5' positions could be compensated by a hydroxy group at position 6 (baicalein). Similar requirements have been reported for flavonoid inhibitors of protein kinase C that act competitively with ATP, suggesting interaction with a conserved protein feature. Formation of the cleavable complex is a cytotoxic lesion that may contribute to the growth-inhibitory properties of flavones observed for three human tumour cell lines. These results are discussed in regard to the selectivity of antiviral agents.
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PMID:Site-specific DNA cleavage by mammalian DNA topoisomerase II induced by novel flavone and catechin derivatives. 131 32

A novobiocin-resistant subline of WEHI-3B D+ murine monomyelocytic leukemia cells was developed by the continuous exposure of cells to this agent in vitro. Sensitive (WEHI-3B/S) and novobiocin-resistant (WEHI-3B/NOVO) sublines were cloned in vitro. WEHI-3B/NOVO cells were stable in the absence of novobiocin for more than 3 months, and the sensitive and resistant clones displayed the same growth rate, cell cycle distribution, cell size, DNA and protein content, and cloning efficiency. Novobiocin has been shown to compete with ATP for the ATP-binding site of topoisomerase II; therefore, intracellular ATP levels can influence the cellular sensitivity to novobiocin. High-performance liquid chromatographic analysis of total cell extracts demonstrated that no difference exists between WEHI-3B/S and WEHI-3B/NOVO cells in the content of ATP. Furthermore, exposure of both cell lines to novobiocin did not affect intracellular ATP levels. In addition to an approximately 2-fold level of resistance to novobiocin, the WEHI-3B/NOVO subline was also 7- and 11-fold cross-resistant to the topoisomerase II-targeted drugs, teniposide and etoposide (VP-16), respectively. A lower level of cross-resistance, comparable to that of novobiocin, was observed in WEHI-3B/NOVO cells for the intercalating topoisomerase II-reactive drugs, doxorubicin, 4'-(9-acridinylamino)methanesulfon-m-anisidide and aclacinomycin A, while the sensitivity to the cytotoxic action of the non-topoisomerase II-acting agents, camptothecin and vincristine, was not altered. After 3-6 h of exposure to 1 microM VP-16, WEHI-3B/S cells accumulated in the S and G2 + M phases of the cell cycle. Similar changes were detected in WEHI-3B/NOVO cells only after exposure to a 10-fold higher concentration of VP-16. Exposure to 150 microM novobiocin caused an accumulation of WEHI-3B/S cells in the G0-G1 phase of the cell cycle but did not affect the cell cycle distribution of WEHI-3B/NOVO cells, while camptothecin induced the same type and extent of changes in the cell cycle distribution of both cell lines. Although the WEHI-3B/NOVO subline appeared to be less responsive to the differentiation-inducing activity of novobiocin and teniposide, the capacity of WEHI-3B/NOVO cells to respond to the differentiation-inducing agent 13-cis-retinoic acid was not significantly different from that of WEHI-3B/S cells. A slight decrease in the accumulation of VP-16 occurred in the resistant cell line, which did not appear to be of sufficient magnitude to account for the 11-fold increase in the degree of resistance to this agent.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Development and characterization of a WEHI-3B D+ monomyelocytic leukemia cell line resistant to novobiocin and cross-resistant to other topoisomerase II-targeted drugs. 131 27

Drosophila melanogaster topoisomerase II is capable of joining phi X174 (+) strand DNA that it has cleaved to duplex oligonucleotide acceptor molecules by an intermolecular ligation reaction (Gale, K. C. and Osheroff, N. (1990) Biochemistry 29, 9538-9545). In order to investigate potential mechanisms for topoisomerase II-mediated DNA recombination, this intrinsic enzyme activity was further characterized. Intermolecular DNA ligation proceeded in a time-dependent fashion and was concentration-dependent with respect to oligonucleotide. The covalent linkage between phi X174 (+) strand DNA and acceptor molecules was confirmed by Southern analysis and alkaline gel electrophoresis. Topoisomerase II-mediated intermolecular DNA ligation required the oligonucleotide to contain a 3'-OH terminus. Moreover, the reaction was dependent on the presence of a divalent cation, was inhibited by salt, and was not affected by the presence of ATP. The enzyme was capable of ligating phi X174 (+) strand DNA to double-stranded oligonucleotides that contained 5'-overhang, 3'-overhand, or blunt ends. Single-stranded, nicked, or gapped oligonucleotides also could be used as acceptor molecules. These results demonstrate that the type II enzyme has an intrinsic ability to mediate illegitimate DNA recombination in vitro and suggests possible roles for topoisomerase II in nucleic acid recombination in vivo.
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PMID:Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination. 131 9

The catalytic activity of topoisomerase II is stimulated approximately 2-3-fold following phosphorylation by casein kinase II (Ackerman, P., Glover, C. V. C., and Osheroff, N. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3164-3168). In order to delineate the mechanism by which the activity of the enzyme is enhanced, the effects of casein kinase II-mediated phosphorylation on the individual steps of the catalytic cycle of Drosophila topoisomerase II were characterized. Phosphorylation did not affect reaction steps that preceded hydrolysis of the enzyme's high energy ATP cofactor. This included enzyme-DNA binding, pre-strand passage DNA cleavage/religation, the double-stranded DNA passage event, and post-strand passage DNA cleavage/religation. In contrast, the rate of topoisomerase II-mediated ATP hydrolysis was stimulated 2.7-fold following phosphorylation by casein kinase II. Since ATP hydrolysis is a prerequisite for enzyme turnover, it is concluded that phosphorylation modulates the overall catalytic activity of topoisomerase II by stimulating the enzyme's ATPase activity.
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PMID:Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis. 132 2

The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable beta,gamma-imido analog of ATP, was carried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNA-bound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex.
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PMID:The capture of a DNA double helix by an ATP-dependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. 133 Mar 27

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).
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PMID:DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes. 133 86

We describe an in vitro system for detection of topoisomerase activity from lysed mitochondria. Mitochondria were isolated from a suspension of cultured Chenopodium album cells. We observed a high activity in relaxation of negatively supercoiled DNA (pBR322). Addition of ATP had no effect on the activity. Topoisomers obtained from negatively supercoiled DNA were identical with topoisomers produced by the topoisomerase I of E. coli. The mitochondrial activity was dependent on the presence of Mg2+ ions and could be inhibited by novobiocin and N-ethylmaleimide. Nalidixic acid and berenil had no influence on the mitochondrial topoisomerase activity. These features characterize the enzyme as a type I topoisomerase.
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PMID:Topoisomerase activity in mitochondrial lysates of a higher plant (Chenopodium album L.). 133 21


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