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 topoisomerase activity was purified from mature ovaries and from nuclei of stage 6 oocytes of Xenopus laevis. From both preparations we obtained a single polypeptide chain having an estimated molecular weight of 67,000. The enzyme purified from ovaries is active in the presence of 150 mM monovalent cation, but its activity is more than 1 order of magnitude higher in the presence of 6 mM Mg2+; the enzyme purified from nuclei requires Mg2+ through all the steps of purification. Enucleated oocytes are devoid of topoisomerase activity but are able to convert the nuclear enzyme to a species active also in the presence of monovalent cations. The difference in ionic requirement between the nuclear topoisomerase and the enzyme purified from ovaries as well as the topoisomerases from other eukaryotic sources, which are most active in the presence of monovalent cations, may depend on the source of the enzyme and/or on the extraction procedure. Ovarian and nuclear topoisomerases catalyze relaxation of both negatively and positively superhelical DNA; the relaxed isomers produced in the presence of Mg2+ have a few positive superhelical turns.
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PMID:Purification and characterization of Xenopus laevis type I topoisomerase. 626 Jul 73

E. coli RecA protein and topoisomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg2+, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When superhelical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages phi X174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of chimeric phage, M13G ori 1, to form catenanes, at least half of which survive heating at 80 degrees C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.
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PMID:Homologous pairing and topological linkage of DNA molecules by combined action of E. coli RecA protein and topoisomerase I. 626 87

The lower eukaryote Ustilago maydis contains a topoisomerase that removes supercoils from negative and positive superhelical DNA. The enzyme may be a multimeric protein or an aggregate of polypeptides with a native molecular weight of 270,000 as estimated by gel filtration. No cofactors are required by the enzyme, but activity is enhanced by Mg2+. Dependence of activity upon enzyme concentration is not linear. Below a threshold level where topoisomerase cannot ordinarily be detected, addition of H1 histone sharply stimulates activity. ATP and a number of structural analogues inhibit the enzyme.
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PMID:Purification and properties of a topoisomerase from Ustilago maydis. 627 Jan 8

We have purified a deoxyribonucleic acid topoisomerase to near homogeneity from the nuclei of mature chicken erythrocytes. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. It is unable to resolve topologically knotted circular duplex DNA. These properties resemble those of type I eukaryotic topoisomerases capable of breaking and rejoining one strand of duplex DNA at a time. The sedimentation value of the protein is 4.4 S. The molecular weight of the reduced, denatured protein is 100K. After elution from sodium dodecyl sulfate (NaDodSO4) gels and renaturation, topoisomerase activity is found in the band at 100K and in minor bands at 95K, 78K, and 73K. The minor bands are likely to be proteolytic fragments since the Mr 100K protein is cleaved by trypsin to fragments of similar or even smaller size with retention of activity. At KCl concentrations suboptimal for the 100K form, the trypsin cleaved form is severalfold more active than the 100K form. Single-stranded DNA, but not duplex DNA or RNA, inhibits DNA relaxing activity, presumably by forming a covalent complex at the enzyme active site. Preincubation of the enzyme with single-stranded DNA leads to the depletion, in NaDodSO4-polyacrylamide gels, of protein bands corresponding to the 100K topoisomerase, its putative proteolytic fragments, and its tryptic fragments. The reaction which leads to band depletion requires active topoisomerase and conditions where single-stranded DNA inhibits relaxing activity. The band depletion technique provides a convenient assay for the polynucleotide binding activity of topoisomerases and possibly other proteins. The function of the enzyme in the inactive nuclei of mature chicken erythrocytes is unclear. The estimated content of chicken erythrocyte topoisomerase per unit DNA is comparable to that in nuclei active in replication and transcription.
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PMID:Topoisomerase I from chicken erythrocytes: purification, characterization, and detection by a deoxyribonucleic acid binding assay. 630 1

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S1 nuclease cleavage. The S1-cleavage occurred once at these sites. In the absence of added Mg2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S1-cleavable, unbasepaired sites.
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PMID:Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease. 630 25

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg2+ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3' to 5' exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.
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PMID:A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus. 630 34

A type I DNA topoisomerase has been isolated from the nuclei of the flagellate Trypanosoma cruzi, using poly(ethylene glycol) fractionation and chromatography on hydroxyapatite and on phosphocellulose. The relaxation activity was ATP-independent, enhanced by Mg2+ and spermidine. The enzyme removed supercoils from negative and positive superhelical DNAs. Topoisomerase activity was associated with a polypeptide of Mr about 65000 as shown by glycerol gradient centrifugation and by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels.
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PMID:A type I DNA topoisomerase from Trypanosoma cruzi. 630 14

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.
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PMID:Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme. 632 14

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
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PMID:Purification and properties of human DNA helicase VI. 754 99

A common strongly ordered multi-step-pattern of endogenous DNA degradation was induced in rat liver nuclei and intact thymocytes, prepared in the presence of chelating agents and incubated in the presence of CaCl2 and/or MgCl2. It consisted of sequential generation of 0.3 Mbp, then 0.05 Mbp DNA fragments and finally of oligo- and mononucleosomal DNA. Oligonucleosomal DNA was generated when the genome had already been disintegrated into 0.05 Mbp DNA fragments. ZnCl2 completely inhibited advanced genome cleavage to oligo- and mononucleosomal DNA without affecting the initial generation of large DNA fragments. Therefore, the endonucleolytic activity which produce large DNA fragments is different from Ca2+/Mg2+ endonuclease. The similar pattern of DNA degradation was observed in thymocytes treated with dexamethasone and with the topoisomerase II inhibitor VM-26, the agents known to induce apoptosis. The effect of VM-26 strongly suggests the involvement of topoisomerase II in generation of large DNA fragments. Multi-level organization and regulation of the chromatin structure determine the stepwise process of genome degradation. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation thus leading to cell death in terminal differentiation or stress-induced apoptosis.
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PMID:Comparative study of induction of endogenous DNA degradation in rat liver nuclei and intact thymocytes. 774 35


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