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)

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.
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PMID:The structure of SV 40 chromatin. 2 73

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.
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PMID:Extracts of Drosophila embryos mediate chromatin assembly in vitro. 11 49

Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of close-circular DNA molecules differing in their number of superhelical turns (tau) around a mean value of tau in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around tau of DNA molecules of different samples overlappped, the difference in the mean number of superhelical turns from sample to sample, delta tau, could be determined and used to measure the mean number (tau) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian-like distribution of molecules differing by integral numbers around a mean value of tau = -24 +/- 2 at 37 degree C [in 0.2 M NaC1, 10 mM Tris-HC1 (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in tau is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phospholiester bond is closed in vivo. When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsC1-propidium diiodide gradient, the number of delta tau = 24 +/- 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28 degree upon intercalation of one molecule of EtdBr.
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PMID:Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. 17 79

The DNA untwisting enzyme has been purified approximately 300-fold from rat liver nuclei. The protein is greater than 90% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis. The native enzyme has a molecular weight between 64 000 and 68 000 and is composed of a single polypeptide chain. Evidence is presented that the protein can act catalytically. A trace amount of endonuclease activity associated with the most pure fraction could be a contaminant or it could be due to the action of the DNA untwisting enzyme itself.
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PMID:Purification and characterization of the DNA untwisting enzyme from rat liver. 18 21

Strands of DNA that have been broken by the DNA untwisting enzyme exhibit a reduced buoyant density in alkaline CsCl due to bound protein. A covalent linkage between the DNA and the enzyme was indicated by the stability of the complex in alkali (pH greaterthan 12.7), in 7 M guanidine-HCl, and at 90 degrees in 1% Sarkosyl for 5 min. The single-strand breaks generated by the enzyme are resistant to exonuclease III, indicating that the protein is attached to one of the ends of the broken strands. The free end of the broken strand bears a 5'-hydroxyl group, indicating attachment of the protein to the 3'-phosphoryl terminus. A nucleotide-peptide linkage involving a phosphoamide bond is unlikely since the complex is resistant to 3.5 M hydroxylamine at pH 4.75.
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PMID:Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. 19 5

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.
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PMID:DNA-relaxing enzyme from Micrococcus luteus. 20 27

Renaturation of two complementary single-stranded circles should be limited by topological constraints against the rewinding of the DNA helix. If a mixture of complementary single-stranded rings is annealed and then treated with the DNA untwisting enzyme, the DNA circles completely renature as judged by (i) the presence of interlocked rings that sediment at 53 S in alkali, (ii) the buoyant density of the renatured DNA in CsCl gradients containing ethidium bromide, and (iii) the resistance of the product to the single-strand-specific S1 nuclease. Therefore, the DNA untwisting enzyme is able to provide a transient single-strand break that is sufficient to allow the two strands to completely rewind. The possibility that the untwisting enzyme might facilitate the initiation of the process of genetic recombination is discussed.
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PMID:Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme. 20 51

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.
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PMID:The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization. 20 80

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.
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PMID:Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex. 20 12

SV40 chromatin can be isolated in two forms: At moderate ionic strength (mu = 0.1-0.3) it contains histone H1 in addition to the four nucleosomal histones and has a highly condensed appearance in the electron microscope, being composed of a few closely connected large spheres [190 A (160, 220) diameter]. At high ionic strength (mu = 0.6-0.8) or after prolonged exposure to very low ionic strengths (mu less than 0.02), the compact form unfolds and the chromatin shows a typical nucleosomal morphology. Native SV40 DNA-protein complexes contain a median number of 24 nucleosomes. The number of superhelical turns does not differ in DNA obtained from the compact and the unfolded forms of chromatin. DNA-relaxing enzyme is found associated with SV40 chromatin and is capable of acting both on extraneously added circular DNA and on its own DNA in the nucleoprotein complex. Purified DNA-relaxing enzyme forms transiently nicked DNA intermediates where the enzyme can be found covalently attached to the site of the nick in the DNA. Transcriptionally active SV40 complexes undergo the same ionic-strength-dependent structural transition as that of bulk SV40 chromatin and may therefore also have a compact configuration at physiological salt concentrations.
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PMID:Biochemical and ultrastructural analysis of SV40 chromatin. 20 38


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