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 DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.
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PMID:A DNA helicase from Xenopus laevis ovaries. 285 68

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.
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PMID:Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells. 302 15

It has been suggested that herpes simplex virus (HSV) type 1 may induce a virus-specific DNA topoisomerase activity which copurifies with virus-induced DNA polymerase. We have examined DNA topoisomerase (TOPO) I and II activities in HSV-2-infected HeLa S3 cells. Both activities were partially purified using DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose column chromatography. It was found that both activities could be separated from HSV-2-specific DNA polymerase. Throughout the purification TOPO I could be immunologically detected with a monoclonal antibody developed against human TOPO I. Regardless of the source, mock- or HSV-2-infected human cells, both types of topoisomerase were equally tolerant of 200 mM-KCl. There appeared to be no apparent heterogeneity of TOPO I in HeLa S3 cells through the course of the HSV-2 infection. We conclude that host cell topoisomerases are quite stable in HSV-2-infected HeLa S3 cells and that there is no evidence that HSV-2 is capable of inducing HSV-2-specific TOPO I and TOPO II activities.
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PMID:Studies on DNA topoisomerases I and II in herpes simplex virus type 2-infected cells. 303 49

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.
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PMID:DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization. 309 38

Several mechanisms are proposed for explaining the antitumor activity and the toxicity of anthracyclines. The first recognized biochemical target is DNA. Anthracyclines and DNA lead to the formation of complexes of intercalation. The intercalation can explain biochemical properties such as inhibition of DNA polymerase and of RNA polymerase. On the other hand, the intercalation cannot explain the chromosomal damages observed in cancer cells following in vivo administration or in vitro incubation. Additional mechanisms are proposed such as biological reduction of quinone C ring, leading to the formation of radical species able to react covalently with DNA. More recently, an interaction of anthracyclines with topoisomerase II has been also described. There is no clear correlation between antitumour efficacy and DNA intercalation. However it must be pointed out that no anthracycline has been found so far which shows antitumour activity dissociated from the ability of interacting with DNA. Anthracyclines interact with membranes: interaction with negatively charged phospholipids like cardiolipin; peroxidation of membrane lipids following biological reduction of the quinone C ring. These membrane effects are believed to be responsible for chronic cardiac toxicity. The clinical activity of daunorubicin and of doxorubicin leads to considerable work with the hope to discover more active and/or less toxic congeners. Several possibilities are investigated: isolation of new anthracyclines from natural sources (fermentation broths); chemical modifications of the whole molecule; total synthesis of new sugars and of new aglycones.
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PMID:[Structure and activity of anthracyclines]. 355 Jun 4

DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly alpha-polymerase as judged from the size determined by sucrose density gradient centrifugation. However, only beta-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topoisomerase.
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PMID:DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers. 609 29

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.
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PMID:DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase. 617 42

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).
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PMID:Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. 629 95

The purified type II DNA topoisomerase from the embryos of Drosophila melanogaster exists in its native form as a dimer of 170,000-dalton polypeptides. In addition to the 170,000-dalton polypeptides, 3 polypeptides with molecular weights of 151,000, 141,000, and 132,000 were resolved when the enzyme was analyzed by electrophoresis under denaturing conditions. All four polypeptides can participate in the topoisomerase cleavage reaction and form covalent complexes with the cleaved DNA. Furthermore, immunochemical and biochemical data showed that they are structurally related and, therefore, the smaller polypeptides are likely generated from the 170,000-dalton polypeptide by proteolysis. The double strand DNA cleavage reaction of Drosophila topoisomerase has different site specificity from the Escherichia coli DNA gyrase-effected reaction. However, they result in an identical DNA structure at the cleavage site, which is a staggered double strand break with 4-nucleotide long 5'-protruding ends. The 3'-ends at the site of cleavage by Drosophila topoisomerase II have free hydroxyl groups and can be extended by exactly 4 nucleotides with T4 DNA polymerase, while the 5'-ends are covalently linked to the topoisomerase molecules. This similarity in cleavage site structure for Drosophila topoisomerase II and E. coli DNA gyrase suggests that they share some fundamental features in their mechanism of action.
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PMID:Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melanogaster. 630 84


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