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)

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 have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
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PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52

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 reported previously that rat liver mitochondria contain a topoisomerase and have shown it to be distinct from the nuclear enzyme by its sensitivity to Berenil and ethidium bromide. We report here some additional characterization. The enzyme differs further from its nuclear counterpart in its failure to bind to ssDNA cellulose and its chromatographic behavior on Sephadex; the latter procedure yields an Mr of 44 000 for the mitochondrial and 70 000 for the nuclear enzyme. The topoisomerase is strongly associated with mitochondrial membranes; only 10% of the activity could be extracted. The pH optimum of the enzyme falls between 6.0 and 8.5, with an NaCl optimum of 0.13 M in 0.1 M Tris (pH 8.3). Dithiothreitol is required, while N-ethylmaleimide is inhibitory. Tosylphenylalanine chloromethyl ketone, a serine proteinase inhibitor, abolishes activity; another, phenylmethanesulfonyl fluoride, has no effect. Berenil, a non-intercalating drug, and four of its analogues all inhibit with up to 100-fold differences in potency. No dependence on ATP, Mg2+, or both together could be shown. Neither novobiocin nor oxolinic acid shows any inhibitory effect. Nicked circles are generated in the presence of DMSO. These three observations are consistent with the topoisomerase being of the Type I class. Positively supercoiled pBR322 DNA, whose 6-8 positive turns were generated by altering solution conditions, is relaxed by the enzyme, indicating a lack of requirement for a negatively supercoiled substrate. We have also examined a partially purified preparation of the corresponding mitochondrial enzyme from mouse L cells. This enzyme is largely similar in properties to the rat liver enzyme. In isolated mitochondria, Berenil causes biphasic alterations in [3H]dATP incorporation into DNA, 10(-4) mM stimulating 2-fold, while higher concentrations inhibit. [3H]UTP incorporation into mitochondrial RNA also follows this pattern.
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PMID:Studies on mitochondrial type I topoisomerase and on its function. 298 52

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.
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PMID:Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits. 299 Dec 31

Type I topoisomerases have been purified from nuclei and mitochondria of human acute lymphoblastic leukemia cells. Both of these ATP-independent enzymes are actually found to be inhibited by ATP at physiologically significant concentrations. Other adenine nucleotides showed varying effects: ADP inhibited only at high concentrations; AMP had no effect on either topoisomerase. Both enzymes were also inhibited by dATP. The importance of the adenine ring structure was confirmed by the lack of an inhibitory effect observed with equivalent levels of GTP, UTP, CTP, or their deoxy counterparts. Assays performed in the presence of nonhydrolyzable analogs of ATP suggest that hydrolysis of ATP does not accompany this enzyme inhibition. This was supported by direct determination of the ATPase activity of the purified enzymes. Type I topoisomerase from calf thymus and HeLa cells were also found to be sensitive to ATP. These results suggest that mammalian type I topoisomerases in general may possess a nucleotide-binding site that may be involved in regulation of enzyme activity.
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PMID:ATP inhibits nuclear and mitochondrial type I topoisomerases from human leukemia cells. 300 64

DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.
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PMID:Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase. Demonstration of an active covalent protein-DNA complex. 301 47

DNA topoisomerase II was purified from calf thymus nuclei by a simple and fast four-step procedure: selective ammonium sulfate precipitation, chromatography on blue-Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on ATP or dATP, the Km values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5-8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaCl. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.
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PMID:Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa. 302 77


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