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Query: UNIPROT:P06889 (Mol)
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Strains of Salmonella typhimurium deficient in topoisomerase I activity (topA mutants) are UV sensitive and non-mutable (Overbye and Margolin: J Bacteriol 146:170-178, 1981). Using lac-operon fusions to DNA damage inducible (din) loci we investigated whether these observations could be explained by an inability of topA strains to efficiently induce DNA damage responses. Mitomycin C (MMC)-induced expression of lac-operon fusions to uvrB and to a second SOS locus, din-9, was largely eliminated in topA bacteria. The inducible expression of several other din-fusions was also diminished. This inducibility defect was mimicked by growth of din-9 topA+ bacteria in media of high osmolarity, a condition that leads to increased DNA supercoiling. Inhibitors of DNA gyrase efficiently induced din-9 in topA bacteria. Together, these results suggest that the topA effect on din expression may be mediated at the level of DNA supercoiling. The sensitivities of a number of din-fusions to topA paralleled the degree to which they were repressed by excess LexA, suggesting that mutations in topA might influence LexA-operator interactions and/or increase lexA expression.
Environ Mol Mutagen 1992
PMID:Mutations in topA interfere with the inducible expression of DNA damage response loci in Salmonella typhimurium. 131 67

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.
Plant Mol Biol 1992 May
PMID:In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells. 132 Apr 23

We investigated the mode of action of the antitumor drug, camptothecin, by use of a partly double-stranded suicide DNA substrate which enables uncoupling of the cleavage and religation half-reactions of topoisomerase I. The suicide DNA substrate contains a single topoisomerase I site at which SDS cleavage is strongly enhanced by camptothecin on normal double-stranded DNA. The results show that the religation reaction of topoisomerase I per se is strongly inhibited at this site compared to site that is only marginally affected by camptothecin on double-stranded DNA. This study hereby directly demonstrates that camptothecin-mediated stability of a topoisomerase I-DNA complex is sequence-dependent. The influence of camptothecin on the suicide cleavage reaction of topoisomerase I was also investigated. Surprisingly, the cleavage reaction per se is strongly inhibited by the drug. However, reformation of a cleavable suicide DNA substrate, which is fully double-stranded downstream from the cleavage position except for a nick, completely reverses the inhibitory effect of the drug on the cleavage reaction. The results suggest that the inhibitory effect of camptothecin on cleavage is due to a general decrease in the noncovalent interaction of topoisomerase I with partly double-stranded suicide DNA substrates. Based on the findings, a plausible model for camptothecin action is discussed.
J Mol Biol 1992 Dec 20
PMID:Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I. 133 13

The relationship between the loss of culturability of Escherichia coli cells in seawater and the DNA supercoiling level of a reporter plasmid (pUC8) have been studied under different experimental conditions. Transfer to seawater of cells grown at low osmolarity decreased their ability to grow without apparent modification of the plasmid supercoiling. We found that E. coli cells could be protected against seawater-induced loss of culturability by increasing their DNA-negative supercoiling in response to environmental factors: either a growth at high osmolarity before the transfer to seawater, or addition of organic matter (50-mg/l peptone) in seawater. We further found conditions where a DNA-induced relaxation was accompanied by an increase in seawater sensitivity. Indeed, inactivation of either one of the subunits A and B of DNA gyrase, which leads to important DNA relaxation, was accompanied in both cases by an increased loss of culturability of conditional mutants after transfer to seawater which could not be explained uniquely by the increase in the temperature required to inactivate the gyrase. Similarly, a strain harbouring a mutation in topoisomerase I, compensated by another mutation in subunit B of the gyrase, was more sensitive to seawater than the isogenic wild-type cell and this greater sensitivity was correlated to a relaxation of plasmid DNA. Again, in these different cases, a previous growth at high osmolarity protected against this seawater sensitivity. We thus propose that the ability of E. coli cells to survive in seawater and maintain their ability to grow on culture media could be linked, at least in part, to the topological state of their DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Ecol 1992 Oct
PMID:Influence of DNA supercoiling on the loss of culturability of Escherichia coli cells incubated in seawater. 134 94

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.
Brain Res Mol Brain Res 1992 Jan
PMID:Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon. 137 74

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded ln DNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA. As cellular uptake of DNA also correlated with the surface binding, these data support electrophoresis of surface bound DNA as the dominating mechanism for the DNA entry. Second, the variations of TE for different topological forms of DNA reflected their relative stability in the host cells. Third, since the loaded DNA could be either rapidly degraded by the host enzyme or expressed, they were unlikely coated with a layer of protective lipid membrane. Thus, PEF induced cellular uptake of DNA is unlikely by the endocytotic mechanisms as was reported previously for the liposomes (Chernomordik, L. V., A. V. Sokolov, and V. G. Budker. 1990.Biochim. Biophys. Acta. 1024:179-183).
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PMID:Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency. 142 Sep 22

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP gamma S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mM-NaCl or 5 mM-ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.
J Mol Biol 1992 Jul 05
PMID:Unwinding of heterologous DNA by RecA protein during the search for homologous sequences. 161 46

Two molecular forms of topoisomerase I differing in size and sensitivity to camptothecin were isolated from calf thymus. Mapping of topo I cleavage sites of the cloned chicken alpha A-globin and human c-Ha-ras genes was carried out. Camptothecin was shown to affect site specificity of the topoisomerases.
Mol Cell Biochem 1991 Mar 13
PMID:Specific cleavage of chicken alpha A-globin and human c-Ha-ras genes by two molecular forms of calf thymus topoisomerase I. 165 Apr 25

We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits eukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.
Plant Mol Biol 1991 Jan
PMID:Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays. 165 30

With the aim of obtaining new inhibitors of topoisomerases, we have evaluated various heterocyclic quinone derivatives for their ability to induce topoisomerase I (Topo I)- or Topo II-associated DNA breaks, using P388 cell nuclear extract. Several compounds belonging to the indolo[3,2-c]quinoline-1,4-dione series have been shown to possess DNA-cleavage activity. Further analysis using purified Topo I and II preparations has indicated that the members of the series stimulate cleavable complex formation of both Topo I and II. 3-Methoxy-11H-pyrido[3',4':4,5]pyrrolo[3,2-c] quinoline-1,4-dione (AzalQD), one of the most active members of the series, stimulates cleavable complex formation and inhibits the catalytic activities of both eukaryotic Topo I and II, with, however, less potency than camptothecin and etoposide. Topo I cleavage site patterns for AzalQD and camptothecin were found to be nearly identical, with, however, some differences in cleavage site intensities. Use of filter binding assays also indicates that AzalQD is at least 10 times more potent against Topo I than against Topo II. Structure-activity relationships of indoloquinolinedione derivatives have been established and have shown that Topo I and II inhibitions are strongly linked, with a dose-selective preference towards Topo I. AzalQD does not display detectable DNA-unwinding properties. AzalQD induces a preferential cytotoxicity for the yeast strain JN2-134 bearing the human top1 gene under the control fo the GAL1 promoter, indicating that Topo I inhibition is responsible for the yeast cytotoxicity. These data indicate that AzalQD and its structural analogs represent a new distinct class of eukaryotic Topo I and II inhibitors.
Mol Pharmacol 1991 Nov
PMID:Inhibition of eukaryotic DNA topoisomerase I and II activities by indoloquinolinedione derivatives. 165 5


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