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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.
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PMID:Chloroplast DNA topoisomerase I from cauliflower. 184 83

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

The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.
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PMID:Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells. 231 56

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

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

The fraction DE-B obtained by fractionating an extract from rat mammary adenocarcinoma cells on a DEAE-Sephadex column was used for transcribing linear and supercoiled rRNA gene (rDNA). This fraction, which is known to contain RNA polymerase I and essential transcription factors, also contains DNA topoisomerase I activity. Inhibition of this topoisomerase activity by the selective inhibitor camptothecin markedly diminished transcription of supercoiled rDNA, and at a concentration of 150 microM, camptothecin almost completely inhibited DNA topoisomerase I activity and supercoiled rDNA transcription. Addition of exogenous calf thymus DNA topoisomerase I to the sample containing the drug restored the ability of the extract to transcribe supercoiled rDNA. Camptothecin, even at a concentration of 500 microM, had no significant effect on the transcription of linear rDNA. These studies show that relaxation of supercoiled rDNA by DNA topoisomerase I is essential for its transcription. The preferential inhibition of rRNA synthesis in vivo following treatment with camptothecin is probably due to selective camptothecin inhibition of DNA topoisomerase I activity.
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PMID:Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene. 303 39

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

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases alpha and delta, topoisomerase II, and replication protein A, (RP-A).
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PMID:Cofractionation of HeLa cell replication proteins with ors-binding activity. 767 29

In a search for factors that influence the process of erythroid differentiation at the molecular level, we have identified UB2, a nuclear protein factor that was originally observed for its ability to bind to a very specific and highly conserved sequence motif present in human, mouse, rabbit, and chicken beta-globin genes, as well as carbonic anhydrase I, c-myb, and the immunoglobulin heavy chain enhancer region. It was also observed for its appearance in undifferentiated but not differentiated mouse erythroleukemia cells. Purification of UB2 by DEAE-cellulose chromatography and repeated passages through a DNA affinity column, revealed a complex pattern with three major components of 170, 116, and 48 kDa, respectively. The 170-kDa protein was identified as topoisomerase (topo) II by Western blot analysis, catalytic assays, and antibody interference with UB2 binding. The complex topo II in UB2, however, has a more stringent sequence requirement for DNA binding than does topo II. The 116-kDa protein has been determined to be a proteolytic product of topo II. The chromosome scaffold protein 2 (135 kDa) copurified with UB2, and anti-scaffold protein 2 serum inhibited UB2 binding to DNA.
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PMID:Purification and characterization of a nuclear DNA-binding factor complex containing topoisomerase II and chromosome scaffold protein 2. 838 2


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