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

The icosahedral shape of the lambda head suggests a 12-subunit structure of the collapsed DNA inside. The internal space of an icosahedron can optimally be filled by 12 geometrical figures each of which is a combination of a cone and more than half of a sphere. Such a pear-like geometrical figure is, in fact, formed spontaneously by DNA collapsed under certain conditions in vitro (Eickbush & Moudrianakis, 1978). It is proposed that a pear-like structure formed by about 4000 bp is the fundamental structural subunit of packaged lambda DNA. A possible arrangement of the 12 subunits inside the phage head relative to the tail is discussed. We hypothesize that lambda DNA is packaged into proheads in its condensed form. A driving force promoting the DNA translocation could be an ATP-dependent activity of a DNA topoisomerase (gpA/gpNu1), which would induce further reduction in the linking number of the already strongly negatively supercoiled DNA by rotation of one DNA strand around the other. The additional strain accumulated at the end of DNA molecule bound by the topoisomerase beyond a critical value would lead to regional collapse of the viral genome into a pear-like structure.
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PMID:A model of lambda DNA arrangement in the viral particle. 293 61

This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single- and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.
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PMID:Inhibition of epipodophyllotoxin cytotoxicity by interference with topoisomerase-mediated DNA cleavage. 299 Apr 88

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

We have found that purified T4 DNA topoisomerase promotes recombination between two phage lambda DNA molecules in an in vitro system. In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro. ATP is not required for this recombination, while oxolinic acid stimulates it. The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of lambda DNA. Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase. A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits. This model is also applicable to the recombination mediated by T4 DNA topoisomerase.
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PMID:Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro. 300 33

The role of DNA topoisomerases in eucaryotic class III gene transcription in vitro has been studied through the use of inhibitory drugs and antisera to DNA topoisomerases I and II. The DNA topoisomerase II inhibitors, novobiocin and coumermycin AI, were found to inhibit transcription of cloned 5S and tRNA genes. Novobiocin acts by interfering with an ATP-requiring step in the pathway to stable preinitiation complex formation. However, it is unlikely that this step reflects the enzymatic action of DNA topoisomerase II since a specific inhibitor of this enzyme (VM-26) and anti-DNA topoisomerase II antibodies fail to inhibit transcription under conditions where topoisomerase II enzymatic activity is inhibited. Similarly, a specific inhibitor of DNA topoisomerase I (camptothecin) and anti-DNA topoisomerase I antibodies fail to inhibit class III gene transcription. These results argue against a role for either DNA topoisomerase in 5S or tRNA gene transcription in vitro.
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PMID:Novobiocin inhibits RNA polymerase III transcription in vitro by a mechanism distinct from DNA topoisomerase II. 300 85

Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage lambda DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage lambda and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.
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PMID:Illegitimate recombination mediated by T4 DNA topoisomerase in vitro. Recombinants between phage and plasmid DNA molecules. 301 75

Indirect end-labelling and the digestion patterns of endogenous and exogenous nucleases were used to analyse chromatin organization along the ribosomal RNA genes of Dictyostelium discoideum cells. A zone just upstream from the 5' end of the coding region was particularly sensitive to endogenous nucleases. In exponentially growing cells, this hypersensitive zone extended from -350 to -1600 bp relative to the transcription start. In sharp contrast, the DNA between 0 and -350 bp was strongly protected. In differentiating cells, in which the ribosomal RNA transcription rate is low, the 5' hypersensitive zone was more diffuse than in exponentially growing cells, and the protected region at the 5' end of the transcribed region was less pronounced. It is known that where DNA topoisomerase is acting on DNA, the addition of sodium dodecyl sulphate will result in cleavage of the DNA and covalent attachment of the enzyme to the cut DNA end. Treatment of nuclei from both exponentially growing cells and differentiating cells with SDS caused double-stranded cleavages at -200 (i.e. within the protected region), at -2200, and at two sites at about -17 kb. A fraction of the cleavage products appeared to be strongly associated with protein. Novobiocin, a DNA topoisomerase II inhibitor, did not inhibit the SDS-induced cleavages in vegetative cells. However, it significantly reduced the extent of nuclease cleavage within the -350 to -1600 bp hypersensitive zone. The possibility is discussed that there are two DNA topoisomerase-like activities on the ribosomal genes. One is site-specific and novobiocin-insensitive. We speculate that the other is responsible for maintaining DNA at the 5' end of the gene in a torsionally strained, nuclease-hypersensitive state.
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PMID:Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes. 301 83

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.
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PMID:Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. 301 47

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.
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PMID:The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. 302 May 13


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