The use of ethidium bromide-circular DNA complexes for the fluorometric analysis of breakage and joining of DNA

Paoletti, Claude; LePecq, Jean-Bernard; Lehman, I. R.

doi:10.1016/0022-2836(71)90282-8

Cited by 83 publications

(25 citation statements)

References 31 publications

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“…The fluorescence method was that used by Paoletti et al [12] to follow the conversion of closed to nicked DNA circles by endonucleases and the reverse reaction catalysed by polynucleotide ligase.…”

Section: Description Of the Assay Methodsmentioning

confidence: 99%

“…The concentration of DNase necessary to convert 30 % of a given concentration of closed PM2 DNA into nicked DNA in 10 min at 37 "C was determined in a pilot experiment with the fluorescence spectrophotometer, according to the procedure of Paoletti et al [12]. The digestion buffer was 10 mM Tris-HC1, pH 7.4, 5 mM MgC12 and 50 mM KC1.…”

Section: Determination Of Superhelix Densitymentioning

confidence: 99%

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Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

Vosberg¹,

Grossman²,

Vinograd³

1975

European Journal of Biochemistry

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A protein, called relaxation protein because of its ability to remove superhelical turns in closedcircular DNA, has been isolated and partially characterized from the nuclei of LA9 mouse and HeLa cells. The purification was facilitated by an assay method, with PM2 DNA, which used the fluorescence enhancement of the intercalating dye ethidium bromide upon binding to the closedcircular DNA. The amount of dye bound depends upon the degree of the superhelix density of the DNA. The relaxation products were analysed by the buoyant separation method in CsCl containing ethidium bromide and were shown to be completely relaxed. The purification resulted in a single band in a dodecylsulfate gel electrophoresis with an apparent molecular weight of 37000. The pH optimum is 7.0 and the optimal salt concentration is 0.2 M NaC1. The relaxation protein removes negative as well as positive supercoils, the latter generated by the interaction of ethidium bromide with closed-circular DNA. Relaxation of positive supercoils results, after removal of the dye, in the formation of molecules with superhelix densities exceeding that of native PM2 DNA (0.054). The highest negative superhelix density observed was -0.098 k 0.001. The corresponding positive superhelix density has been calculated to be + 0.023.A nicking -swivelling -closing mechanism is postulated, but nicked intermediates have so far not been demonstrated. The relaxation protein is not inhibited by known mammalian endonuclease I inhibitors, except for denatured DNA, and does not possess a conventional polynucleotide ligase activity. The relaxation activity was found to be predominantly in the nuclei, with only small amounts present in the cytoplasm and mitochondria. The biological function of transient swivels induced by the relaxation protein is not known. However, transient swivels are considered necessary or useful in the replication of closed-circular DNA or long linear DNA, respectively. Relaxation protein could replace the combined action of an endonuclease and a ligase ahead of the replication fork. Alternatively, transient swivels could be involved in the transcription process.In 1971 Wang [l] isolated and extensively purified a protein called o from uninfected Escherichiu coli cells. This protein was able to remove supercoils from negatively supercoiled DNA by reducing the topological winding number, a process which requires that the polynucleotide chain be nicked and then be rejoined after swivelling has occurred. Since the reaction did not occur with positively supercoiled DNA, it seemed doubtful that the co protein could replace the combined actions of an endonuclease and a polynucleotide ligase at the growing fork of duplex DNA during DNA replication. Such an action would reduce the length of duplex DNA that rotates aheadEnzymes. E. coli alkaline phosphatase (EC 3.1.3.1); pancreatic DNase (EC 3.1.4.5). of the growing fork during semi-conservative replication.Champoux and Dulbecco [2] in 1972, found a similar relaxation activity in sonicated nuclei of embryonic ...

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Section: Description Of the Assay Methodsmentioning

confidence: 99%

Section: Determination Of Superhelix Densitymentioning

confidence: 99%

Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

Vosberg¹,

Grossman²,

Vinograd³

1975

European Journal of Biochemistry

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“…EB is one of the most sensitive fluorescence probes having a planar structure that binds to DNA in an intercalative fashion [52,53]. The fluorescence of EB increases after intercalating to DNA.…”

Section: Competitive Binding Between Eb and Copper Complexes For Ct-dnamentioning

confidence: 99%

Studies on the interaction of copper complexes of (−)-epicatechin gallate and (−)-epigallocatechin gallate with calf thymus DNA

Ghosh

Sahoo

Jana

et al. 2008

Journal of Inorganic Biochemistry

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“…It was then placed horizontally on top of a sodium dodecyl sulfate/polyacrylamide (5%) slab, which had been cast in such a way that protein markers could be run in two slots flanking the cylindrical gel. Ethidium fluorescence assay of the degree of superhelicity of a covalently closed DNA (14) was done according to the high-pH procedure of fraction VIa protein at 1.6 ,g/ml, and fraction VIb protein at 2.4 pg/ml was incubated at 230. Aliquots (50 ,gl each) were taken out at time intervals and each was mixed with 1.2 ml of a solution containing 20 mM K3PO4 (pH -12), ethidium bromide at 0.5 ,g/ml, and 3 mM EDTA.…”

mentioning

confidence: 99%

Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

Liu¹,

Wang²

1978

Proc. Natl. Acad. Sci. U.S.A.

198

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ABSTRACr Two active components a and P of Micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits litte DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components a and f, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.A novel Escherichia coil enzyme, termed DNA gyrase, has been discovered recently (1). This enzyme converts a covalently closed circular DNA without superhelical turns to a highly negatively supercoiled form. In other words, the enzyme causes a reduction of the linking number (2) of the covalently closed circular duplex. The reaction requires ATP, the hydrolysis of which presumably provides the driving force for the otherwise energetically unfavorable reaction. The ATP-dependent negative supercoiling of DNA promoted by gyrase is phenomenologically opposite to the ATP-independent removal of negative superhelical turns by E. coli w protein [Eco DNA topoisomerase I (3)].* In vitro and in vivo studies indicate that in E. colt two widely separate genetic loci are involved in gyrase activity. These are the locus cou, which determines resistance to two related antibiotics (coumermycin A and novobiocin), and the locus nalA, which determines resistance to another two related drugs (nalidixic and oxolinic acids) (4-6). Gyrase purified from cells sensitive to these antibacterial agents is inhibited by them in its supercoiling of DNA, whereas the same enzyme purified from mutant cells resistant to these agents is not (4-6). Because in vivo these drugs block DNA replication (7-9) and the supercoiling of superinfected X DNA (4-6), the identification of gyrase as the target of these drugs in turn indicates that gyrase is involved in DNA replication and supercoiling in E. coli (1,4,6).E. coli gyrase has been purified by a number of groups (1,5,6,10). The nalA gene product has been purified to homogeneity (5). It appears to have a DNA topoisomerase activity distinct from Eco DNA topoisomerase 1 (5).In this communication, we report our studies on two active components, a and fl, of Micrococcus luteus gyrase. The possibility that negative superhelical turns in a DNA...

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The use of ethidium bromide-circular DNA complexes for the fluorometric analysis of breakage and joining of DNA

Cited by 83 publications

References 31 publications

Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

Studies on the interaction of copper complexes of (−)-epicatechin gallate and (−)-epigallocatechin gallate with calf thymus DNA

Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

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