Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions

We have examined binding properties of and dissociation induced by the intercalating dye ethidium bromide when it interacts with the nucleosome core particle under low ionic strength conditions. Ethidium binding to the core particle results in a reversible dissociation which requires the critical binding of 14 ethidium molecules. Under low ionic strength conditions, dissociation is about 90% completed in 5 h. The observed ethidium binding isotherm was corrected for the presence of free DNA due to particle dissociation. The corrected curve reveals that the binding of ethidium to the core particle itself is a highly cooperative process characterized by a low intrinsic binding constant of KA = 2.4 X 10(4) M-1 and a cooperativity parameter of omega = approximately 140. The number of base pairs excluded to another dye molecule by each bound dye molecule (n) is 4.5. Through the use of a chemical probe, methidiumpropyl-EDTA (MPE), we have localized the initial binding sites of ethidium in the core particle to consist of an average of 27 +/- 4 bp of DNA that are distributed near both ends of the DNA termini. MPE footprint analysis has also revealed that, prior to dissociation, the fractional population of core particles which bind the dye (f) may be as low as 50%. Comparison of the binding and dissociation data showed that the cooperative maximum of the binding curve occurred at or near the critical value, i.e., at the point where dissociation began. The data were used to generate a detailed model for the association of ethidium with chromatin at the level of the nucleosome.     

Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 2. The torsional motion of the DNA is highly constrained and sensitive to pH

The effects of pH on the torsional flexibility of DNA bound to nucleosome core particles were investigated by using time-resolved fluorescence anisotropy decays of intercalated ethidium. The decays were collected by using time-resolved single-photon counting and were fit to a model developed by J. M. Schurr [(1984) Chem. Phys. 84, 71-96] with a nonlinear least-squares-fitting algorithm developed for this purpose. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. Because we see large increases in DNA flexibility and in the rotational diffusion coefficient at binding ratios of 0.6 ethidium/core particle and above, we conclude that, under these conditions, the DNA begins to detach from the protein. At lower binding ratios, we observe only small changes in the anisotropy decay. The torsional parameters obtained are a function of N, the number of base pairs of DNA between points of attachment to the histone core. Only if N is greater than 30 base pairs is the torsional rigidity of DNA on a nucleosome core particle higher than that for DNA free in solution. Also, for reasonable values of N (less than 30), the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the region of the DNA to which the ethidium binds is highly constrained in its motions. pH changes nearly neutrality at moderate ionic strengths (100 mM) have a substantial effect on the fluorescence anisotropy decays, particularly at early times. These analyses indicated that the observed change on increasing pH can be attributed either to a loosening of the contacts between the DNA and the histone core (increasing N) or to a substantial relaxing of the torsional rigidity of the DNA.     

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.     

Studies on amikhellin: I. Intercalative binding to double-stranded DNA

The present paper reports that amikhellin, a drug so far used as a coronary vasodilator, binds to double-stranded DNA by an intercalation process which does not depend upon DNA base composition. The binding to DNA was established by spectrophotometry, ultracentrifugation and competition with ethidium bromide. The parameters of the binding equilibrium were calculated by these two latter methods. Evidence for intercalation was obtained from the observation by viscosimetric experiments of the length increase of sonicated calf thymus DNA and of the untwisting of circular PM2 DNA. The unwinding angle was measured to be 6° per bound drug molecule.     

Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess Eb a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present; otherwise a variable correction factor is required to account for unbound Eb.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

Synthesis, characterization, and DNA binding studies of some mixed-ligand platinum(II) complexes of 1,10-phenanthroline and amino acids

A series of complexes of the type [Pt(phen)(AA)]+ (where AA is the anion of glycine, L-alanine, L-leucine, L-phenylalanine, L-tyrosine, or L-tryptophan) has been synthesized. These complexes have been characterized by electronic absorption, infrared, and 1H NMR spectroscopy. The interaction of these complexes with calf thymus DNA has been studied using fluorescence spectroscopy. They inhibit the intercalation of ethidium bromide in DNA by intercalative binding at low concentrations and show nonintercalative binding at higher concentrations.     

Bifunctional intercalation of antitumor antibiotics BBM-928A and echinomycin with deoxyribonucleic acid. Effects of intercalation on deoxyribonucleic acid degradative activity of bleomycin and phleomycin

The binding of peptide antitumor antibiotics, BBM-928A and echinomycin, to superhelical PM2 DNA and the effects of the resulting conformational changes of DNA on the DNA-degradative activity of two related antitumor antibiotics, bleomycin A2 and phleomycin D1, have been studied. The bifunctional intercalative mode of the DNA binding of BBM-928A concluded previously from viscometric and fluorometric studies has been confirmed by gel electrophoretic analysis. Under the employed electrophoretic conditions, DNA-bound BBM-928A showed little dissociation whereas echinomycin and ethidium bromide showed partial and nearly complete dissociation, respectively. BBM-928A induced neither single-strand nor double-strand breaks in DNA. Competitive binding studies by fluorescence changes suggested that binding sites on DNA molecules for BBM-928A (or echinomycin) may differ from those for ethidium bromide, since binding to DNA by the two drugs was not competitive even at saturating concentrations. The lack of such a competition between the two drugs is not consistent with the neighbor-exclusion principle. The DNA-degradative activity of both bleomycin A2 and phleomycin D1 increased with the removal of the negative superhelicity of DNA by the BBM-928A intercalation and decreased with the formation of positive superhelical turns induced by high concentrations of BBM-928A. Thus the degradative activity of both bleomycin A2 and phleomycin D1 is sensitive in a similar manner to the degree of superhelicity rather than the double helicity of DNA, although there are differences between these two drugs in interaction with DNA.     

Ethidium bromide binding to core particle: comparison with native chromatin.

Ethidium bromide intercalation into DNA of nuclease digested erythrocyte chromatin and core particle, was followed at low ionic strength by fluorescence measurements, equilibrium dialysis using 14C labelled dye, circular dichroism and electron microscopy. High affinity binding sites in the chromatin are no more present in the core particle, i.e. when the linker is removed. In the case of core particle, a cooperative process occurs, accompanied by a partial stripping of the DNA from the core histone. Finally two populations of core particles can be detected by electron microscopy as far as their binding properties are concerned.     

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

Structure of the complex between lac repressor headpiece and operator DNA from measurements of the orientation relaxation and the electric dichroism.

The complex between lac repressor headpiece and short rodlike DNA fragments containing the lac operator sequence is characterised by measurements of the rotation diffusion. Using the method of electric dichroism we measure the rotation relaxation and determine changes in the length of the DNA upon ligand binding with high accuracy. According to these measurements any change in the length of the operator DNA upon binding of the first two headpiece molecules remains below 1A; the electric dichroism also remains virtually unchanged. At high degrees of (unspecific) binding we observe an increase in the rotation relaxation time, which is attributed to an increase of the apparent mean radius of the complex. As a control of our procedure for the determination of length changes we use the intercalation of ethidium bromide and arrive at an increase of the DNA length per bound ethidium of 3.2A (at 3.4A rise per base pair). The results obtained for the headpiece operator complex are not consistent with models assuming large changes of the DNA structure or intercalation of tyrosine residues.     

A study of the interaction between ethidium bromide and rye chromatin: comparison with calf thymus chromatin.

We studied the interaction of ethidium bromide with rye and calf thymus chromatin. Both types of chromatin have the same dye accessibility, which is about 50% of that of DNA. From this result we conclude that the molecular structure of these two chromatins is similar. For rye, the extraction of H1 produces no change in the binding of ethidium bromide. The subsequent extraction of H2A and H2B produces a 14% increase in the binding, and the removal of H3 and H4, another 54% increase. At this stage, the number of binding sites is still less than that of DNA. This is presumably due to the presence of some tightly bound non-histones. Thus, the arginine-rich histones and the tightly bound non-histones are most responsible for limiting the binding of ethidium bromide to rye chromatin.     

Studies of the Fluorophore Sempervirene and its Complexes with DNA

Evidence that the fluorophore sempervirene binds to nucleic acids is presented. The complexes were studied by fluorescence intensity, spectra, decay lifetime, and polarization methods. Both fluorescent and nonfluorescent complexes are formed. The sempervirene is rigidly fixed to DNA. If ethidium bromide and sempervirene are bound to DNA, energy can be transferred from sempervirene to ethidium. Sempervirene is taken up by mammalian cells and appears in the cytoplasm. This unusual new probe should be useful in molecular and cellular investigations.     

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of lambda phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of alpha 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.     

Fluorescence Polarization Studies of the Binding of BMS 181176 to DNA

Abstract

The DNA binding of BMS 181176, an antitumor antibiotic derivative of rebeccamycin was characterized by DNA unwinding assays, as well as by fluorescence emission and polarization spectroscopic techniques. Unwinding and rewinding of supercoiled DNA was interpreted in terms of intercalation of BMS 181176 into DNA BMS 181176 shows an enhanced fluorescence emission upon binding to the AT sequence and no enhancement upon binding to the GC sequence. BMS 181176 appears to be a weaker binder to poly(dAdT).poly(dAdT) compared to doxorubicin and ethidium bromide. When bound to DNA, the rotational motion of BMS 181176 is substantially decreased as evident from the increase in fluorescence polarization. BMS 181176 exhibits a range of binding strengths depending on the DNA This is demonstrated by the Acridine Orange displacement assay using fluorescence polarization.     

Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters

Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.     

Binding of ethidium and bis(methidium)spermine to Z DNA by intercalation

The interaction of ethidium bromide, a DNA intercalating drug, and bis( methidium )spermine, a DNA bis-intercalating compound, with the left-handed Z form of poly(dG-dC) has been studied in 4.4 M NaCl. Spectrophotometric analysis using absorption, fluorescence and circular dichroism indicates that the complex formed between ethidium and Z DNA resembles very closely that formed with B DNA. This suggests that ethidium binds to Z DNA by intercalation. 31P NMR spectra are presented showing both the conversion of the Z form to the B form with increasing amounts of drug and the typical Z form spectrum at low binding densities. Data are also presented which show that the bifunctional intercalator bis( methidium )spermine binds to Z DNA in a manner similar to its binding to B DNA, i.e., by bis-intercalation. These results are important for our understanding the behavior of Z DNA and its biological significance.     

Efficient calf thymus DNA condensation upon binding with novel bile acid polyamine amides

Polyamine amides have been prepared from lithocholic and cholic acids (5beta-colanes) by acylation of tri-Boc-protected tetraamines spermine and thermine. These designed ligands for DNA are polyammonium ions at physiological pH. In NMR spectra, they display 14N-1H 1J = 51 Hz, 1:1:1 triplets, due to the symmetry of the R14NH(3)+ cations. The binding affinities of these conjugates for calf thymus DNA were determined using an ethidium bromide fluorescence quenching assay and compared with spermine and polylysine. DNA-binding affinities were dependent upon both salt concentration and the hydrophobicity or intermolecular bonding (facial effects) of the lipid moieties in these conjugates. Light scattering at 320 nm was used to determine DNA condensation and particle formation. The observed self-assembly phenomena are discussed with respect to DNA charge neutralization and DNA bending with loss of ethidium cation intercalation sites, ultimately leading to DNA condensation. These polyamine amides are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.