Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.     

The EcoRI restriction endonuclease, covalently closed DNA and ethidium bromide.

The reactions of the EcoRI restriction endonuclease on the covalently closed DNA of plasmid pMB9 were studied in the presence of ethidium bromide. At the concentrations of ethidium bromide tested, which covered the range over which the DNA is changed from negatively to positively supercoiled, the dye caused no alteration to the rate at which this enzyme cleaved the covalently closed DNA to yield the open-circle form, but the rate at which these open circles were cleaved to the linear product could be inhibited. The fluorescence change, caused by ethidium bromide binding with different stoichiometries to covalently closed and open-circle DNA, provided a direct and sensitive signal for monitoring the cleavage of DNA by this enzyme. This method was used for a steady-state kinetic analysis of the reaction catalysed by the EcoRI restriction enzyme. Reaction mechanisms where a complex between DNA and Mg2+ is the substrate for this enzyme were eliminated, and instead DNA and Mg2+ must bind to the enzyme in separate stages. The requisite controls for this fluorimetric assay in both steady-state and transient kinetics studies, and its application to other enzymes that alter the structure of covalently closed DNA, are described.     

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.     

A clarification of the complex spectrum observed with the ultraviolet circular dichroism of ethidium bromide bound to DNA.

Ethidium bromide intercalation strongly effects the circular dichroism spectrum of DNA in the region of 230-300 mu, in a complex manner. In this report we present a study that quantitizes the relationships of the circular dichroism spectrum in the region of 230-300 mu and the ethidium bromide induced optical activity centered around 308 mu. We present evidence of two hidden cooperative bands that are probably the negative counterparts of the 308 mu band and 330 mu shoulder positive cooperative bands. The hidden band is quantitatively characterized. We confirm that the direct effect of ethidium bromide on the DNA spectrum is simply linearly proportional to the amount of intercalated dye. We also observe that the ethidium bromide enters freely when there is a molecule intercalated for every 3 sites, but that the intercalation is more difficult when the molecule intercalates at every second site.     

SV40 DNA: quantitative conversion of closed circular to open circular form by an ethidium bromide-restricted endonuclease

Closed circular molecules of SV40 DNA can be quantitatively converted to the open circular form by digestion with endonucleases in the presence of ethidium bromide. Under these conditions almost no molecules were digested beyond the open circular form, whereas in the absence of the dye the DNA was completely fragmented.     

Improved electrophoretic separation of supercoiled and relaxed DNA in the presence of ethidium bromide.

Supercoiled and relaxed DNA were resolved electrophoretically in the presence of 0.5 micrograms/ml ethidium bromide. Under these conditions the Gaussian distributions of topological isomers of both supercoiled and relaxed DNA migrated as discrete bands. The separation of these DNAs was optimized by varying the concentration of electrode buffer. Electrophoresis in the presence of 160 mM Tris-acetate, pH 8.3, 4 mM EDTA resulted in a 20-fold increase in the separation of relaxed and supercoiled DNA relative to electrophoresis in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA.     

Connection of ethidium bromide with single-stranded DNA

The interaction of ethidium bromide with single-stranded synthetic and natural polynucleotides at high temperatures (t = 70 degrees C) and low pH values (pH 3.0) was studied. The isotherms of adsorption of ethidium bromide on single-stranded DNA were obtained. Two modes of binding of single-stranded DNA, strong and weak, were revealed. The values of the corresponding constants of interaction of this ligand and the number of bases per one binding site were determined.     

Interaction of ethidium bromide with DNA as studied by kinetic spectrophotometry.

The interaction of ethidium bromide (EB) with DNA has been investigated using the pulse radiolysis technique. In particular, the absolute rate constant for the reaction of hydrated electrons, generated by single pulses of high-energy electrons, with EB is shown to drop dramatically in the presence of DNA. This drop in diffusion-limited reactivity results from the interaction of EB with DNA, effectively immobilising it, thus lowering the reaction cross-section or probability. Analysis of the resulting kinetic spectrophotometric data shows that they are consistent with a reversible interaction of EB with DNA as described by the law of mass action. The Scatchard-type plots obtained are linear, and give quantitative information on the extent and degree of association, comparable with that obtained by more conventional methods. The potential of the pulse radiolysis technique for studying different types of interactions between small molecules and various biopolymers has been demonstrated.     

De-intercalation of ethidium bromide and propidium iodine from DNA in the presence of caffeine

Caffeine (CAF) is capable of interacting directly with several genotoxic aromatic ligands by stacking aggregation. Formation of such hetero-complexes may diminish pharmacological activity of these ligands, which is often related to its direct interaction with DNA. To check these interactions we performed three independent series of spectroscopic titrations for each ligand (ethidium bromide, EB, and propidium iodine, PI) according to the following setup: DNA with ligand, ligand with CAF and DNA-ligand mixture with CAF. We analyzed DNA-ligand and ligand-CAF mixtures numerically using well known models: McGhee-von Hippel model for ligand-DNA interactions and thermodynamic-statistical model of mixed association of caffeine with aromatic ligands developed by Zdunek et al. (2000). Based on these models we calculated association constants and concentrations of mixture components using a novel method developed here. Results are in good agreement with parameters calculated in separate experiments and demonstrate de-intercalation of EB and PI molecules from DNA caused by CAF.     

Interaction of the dye ethidium bromide with DNA containing guanine repeats.

DNA containing one or more copies of the motifs repeated in telomere sequences has unusual conformational properties. The isolated sequence from the protozoan Oxytricha, dT4G4 has the potential to form tetramers in the presence of sodium or potassium ions. We report here that these tetramers bind ethidium tightly, with an interaction that fulfills several criteria for an intercalative mechanism in the G sequence. By contrast, the 4-fold tandem repeat of this subunit, d(T4G4)4, does not interact specifically with ethidium in the presence of Na+. This difference might have a simple structural basis: the tetramer of dT4G4 forms a stack of four G-quartets in the presence of Na+ or K+, whereas the constraint imposed by the T4 "tethers" in the repeat d(T4G4)4 allows only two layers to form in the presence of Na+. In the presence of sufficient K+, the latter can partially form a four-layer G-quartet structure, which interacts with ethidium. This idea is supported by analysis of a "relaxed" sequence, dT4G4(T7G4)3, which allows formation of four G-quartets and binds ethidium in the presence of Na+ as well as K+. Ethidium (and intercalators generally) should thus be able to retard or inhibit the action of telomerase in the presence of K+.     

Study of complexes of ethidium bromide with DNA by differential pulse voltammetry

The interaction of ethidium bromide with calf thymus DNA was investigated by the method of differential pulse voltammetry. It was found that ethidium bromide binds with DNA in several ways. Corresponding values of the constants and the number of binding sites were determined. The intercalation, semi-intercalation, and electrostatic mechanisms of interaction were shown. The results obtained are in good agreement with the data obtained by spectroscopic (absorption and fluorimetric) methods.     

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.