The fluorescence anisotropy decay due to energy transfers occuring in the ethidium bromide-DNA complex. Determination of the deformation angle of the DNA helix

It has been shown in a preceding work that the fluorescence anisotropy decay of ethidium bromide-DNA complex is accelerated by energy migration between dyes bound to the same DNA molecule. In the present work, this result is confirmed. A quantitative analysis has been performed in the following way. The spectroscopic term of the transfer rate constant has been accurately reevaluated by quantum yield and spectral measurements. One assumes that the dye intercalates between two adjacent base pairs and that its distribution is random along the DNA molecule. One introduces the deformation angle δ of the DNA helix induced by the ethidium bromide intercalation. For several values of δ, the energy migration contribution to the anisotropy decay is computed by a Monte Carlo method. In multiplying these computed functions by the measured brownian anisotropy, one obtains the anisotropy decay curve. Comparison with the experimental data leads to the conclusion that the ethidium bromide molecule unwinds the DNA helix by an angle δ = ?16°. This result is m agreement with the work of other authors. We think that the method used here may provide accurate information on the spatial distribution of an array of chromophores bound to a rigid structure.     

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

Dye titrations of covalently closed supercoiled DNA analyzed by agarose gel electrophoresis.

We have developed a rapid electrophoretic technique for performing ethidium bromide dye titrations in cylindrical 0.7% agarose gels. The technique was used to analyze the extent of supercoiling in circular covalently closed SV40, Co1E1, and pSC101 DNA. We have estimated the superhelical densities of SV40, Co1E1, and pSC101 DNA to be ?0.050, ?0.078, and ?0.085 respectively. The results obtained for native SV40 DNA correlate well with previously published values for the superhelical density of this DNA when these values are corrected to reflect a 26° duplex unwinding angle for ethidium bromide. Ethidium bromide concentrations sufficient to partially relax a supercoiled DNA allow the DNA to be resolved into a series of discrete bands in agarose gels. The distribution of bands represents a natural heterogeneity in the superhelical densities of the DNA molecules in the population.     

Interactions of norharman and harman with DNA.

The interactions of norharman (9H-pyrido [3,4-b] indole) and harman (1-methyl-9H-pyrido [3,4-b] indole) with DNA were studied. DNA caused remarkable fluorescence quenching and change in the absorption spectra of the dyes. Scatchard plots obtained by optical titration gave Kd values of 2.2 X 10(-5)M and 7.7 X 10(-6)M, and apparent numbers of binding sites of 0.13/base and 0.12/base for norharman and harman, respectively. Agarose gel electrophoresis of circular DNA, closed in the presence or absence of norharman revealed that the dye intercalates DNA, thereby causing 17 +/- 3 degrees unwinding of the double helix.     

Interactions of 4'', 6-diamidine-2-phenylindole with synthetic polynucleotides.

4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs. With the exception of the four-stranded helical poly rI which exhibits considerable fluorescence enhancement upon binding of the ligand, none of the single- or multi- stranded polyribonucleotides and ribo-deoxyribonucleotide hybrid structures form fluorescent complexes with the dye. Poly rI is the only RNA which forms a DNA B-like structure (Arnott et al. (1974) Biochem. J. 141, 537). The B conformation of the helix and the absence of guanine appear to be the major determinants of the specificity of the fluorescent binding mode of the dye. Nonfluorescent interactions of the dye with polynucleotides are nonspecific; UV absorption and circular dichroic spectra demonstrate binding to synthetic single- and double-stranded DNA and RNA analogs, including those containing GC base pairs.     

The response of DNA length and twist to changes in ionic strength

We examine twist‐stretch coupling of unconstrained DNA using polyelectrolyte theory as applied to a line‐charge model along with published data on the ionic‐strength dependence of the twist angle. We conclude that twist‐stretch coupling is negative: environmental changes that stretch free DNA, unconstrained by externally applied pulling or twisting forces, are accompanied by unwinding of the double helix. We also analyze a helical model and conclude that the observed unwinding of the DNA helix when ionic strength is decreased is driven by radial swelling of the helix. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 223–226, 2015.     

A theory for polarized fluorescence microscopy of chromosome structures

A theory for the determination of DNA arrangements in DNA-containing specimens, using planar aromatic dye molecules as probes for plane polarization of fluorescence, has been described. At low dye-to-DNA concentrations, the dye molecules are sandwiched between the stacked bases of DNA; hence, the fluorescence from the dye bound to a local region of DNA helix is plane-polarized with the polarization direction perpendicular to the local axis of DNA. The degree of such polarization from an aligned DNA-specimen complexed with dye is determined both by the DNA orientation and the conformational state (e.g., base tilt) of DNA into that specimen. Analysis has been made of the relationship between the degree of polarization and the orientation of the emitting dipoles of dye. The dye complexes may be aligned in a mechanical shear or electric field. However, any change in the orientation distribution of the emitting dipoles due to force fields should be taken into account. With some assumptions and approximations, the magnitude and the direction of maximum polarization can be related to different orders of DNA coiling and to their various combinations. Since the measured polarization is averaged over all DNA regions of the specimen, if the magnitude of polarization is appreciable and the polarization occurs in the specific direction of the specimen, the theory helps to eliminate several probable arrangements of DNA. The predominant molecular features of the actual DNA arrangement can be determined through this process of elimination, as explained in two subsequent papers with T-even bacteriophage and chromosome systems.     

Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association.

Isotherms describing the binding of hydroxystilbamidine to DNA and polydeoxyribonucleotides were obtained by means of sedimentation or dialysis experiments and fluorescence measurements, over a large range of ionic strengths, temperatures and base compositions. Two different sets of binding sites are necessary to explain the shapes of the isotherms. The first one is characterized by a higher binding constant, a topological specificity for the A-T pair, exclusion of four base pairs per bound dye molecule, the involvement of two ion-pairs, an almost purely entropic free energy of binding and a large enhancement of the blue fluorescence (450 nm) when the site corresponds to three adjacent A-T pairs. The latter does not present any specificity nor enhancement of fluorescence and only one ion-pair is formed. From the geometry of the dye and its selective binding to a double stranded structure, the hydroxystilbamidine molecule in the first set of sites is likely to be situated in the small groove astride the two complementary strands and slightly distorting the helical structure. The angle of the dye axis with the helix axis has a value close to 47 degrees. No definite explanation could be given for the specific binding of hydroxystilbamidine but the phenolic hydroxyl group is likely to play a major role. The hydroxystilbamidine molecule can be considered as a useful tool for checking the accessibility of the small groove.     

The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients

A series of covalently closed bacteriophage PM2 DNA samples with varying degrees of superhelicity were prepared in vitro. The amount of bound ethidium per DNA nueleotide needed for the removal of all superhelical turns, v_c⁰, was determined for each sample by a number of methods. In order to evaluate the unwinding angle for the binding of one ethidium molecule to a DNA double helix, the pH dependence of the buoyant densities in CsCI of these samples was examined. A new calibration relating the change in buoyant density of a DNA to the fraction of bases titrated has been obtained, by measuring the buoyant densities of a number of catenanes (interlocked rings) containing both single-stranded and double-stranded λ DNA rings, at a pH such that the single-stranded DNA is fully titrated while the double-stranded DNA is not titrated. This calibration was used to obtain the pH dependence of the fraction of DNA bases titrated for the phage PM2 DNAs with differing extents of supercoiling. A simple theoretical analysis shows that in a restricted pH range close to pH_m, the melting pH of the DNA in the absence of the topological constraint associated with covalently closed double-stranded DNAs, the difference in the fraction of bases titrated at a certain pH between two covalently closed DNAs with different degrees of superhelicity is directly proportional to the difference in the v_c⁰ values of the DNAs. The unwinding angle per bound ethidium molecule can be obtained from the proportionality constant. In this way, it is not necessary to know precisely the actual pH value for either DNA, pH_e, at which the DNA is titrated to the extent that it contains no superhelical turns. The conclusion of the theoretical analysis and the experimental results is that the binding of an ethidium molecule to a double-stranded DNA unwinds the DNA helix by an angle φ_e = 26 °. The uncertainty in this value is estimated to be less than 10%. The new value for φ_e is approximately a factor of two larger than the value 12 °, which has been in use in the past decade. In the earlier alkaline titration results for polyoma DNA (Vinograd et al., 1968), which had been interpreted as supporting the 12 ° value, the calculation of φ_e was critically dependent on knowing pH_e. It is believed that pH_e was underestimated in the earlier work, resulting in a low φ_e value. Since the previous value φ_e = 12 ° has been widely used in the determination of the number of superhelical turns for many DNAs, and in measurements on the angular alterations of the DNA helix by the binding of a variety of small and large molecules and by solvent and temperature changes, the new value φ_e = 26 ° requires proportional adjustments of many previous results.     

The interaction of plant alkaloids with DNA. II. Berberinium chloride.

The interaction of berberinium chloride with DNA has been investigated using spectrophotometry, viscometric titrations with sonicated and closed circular superhelical DNA, and flow polarized fluorescence. The binding results for berberinium were found to fit the neighbor exclusion model. The two viscometric titrations and flow polarized fluorescence results exclusion model. The two viscometric titrations and flow polarized fluorescence results also indicated that berberinium binds to DNA by intercalation. Titration of sonicated DNA with berberinium produced viscosity increases which were less than those obtained with quinacrine and the titration of superhelical DNA indicated a significantly smaller unwinding angle for intercalation of berberinium than for quinacrine. These results can be interpreted in terms of a model in which (i) berberinium is partially intercalated into the double helix, or (ii) the alkaloid is more completely intercalated into the double helix, but causes bending of the helix due to the slight nonplanarity of the berberinium ring system, or (iii) a combination of (i) and (22).     

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding.

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.     

The conformations and energetics of photodamaged DNA

Energy minimization techniques are used in conjunction with the results of small molecule crystallographic studies on relevant compounds to propose structural models for photodamaged DNAs. Specifically, we present models both for a DNA molecule containing a psoralen photo-crosslink and for a DNA molecule containing a thymine photodimer. In both models, significant distortions of the nucleic acid helix are observed, including kinking and unwinding at the damage site and numerous changes in the backbone torsion angles relative to their standard conformations. Both the torsion angle geometries and the energetics of the models are presented in detail.     

The effect of ionic strength on DNA-ligand unwinding angles for acridine and quinoline derivatives.

We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a control for determining superhelix densities at different ionic strengths. Relative to ethidium, 9-aminoacridine and quinacrine had an essentially constant unwinding angle of approximately 17 degrees at all ionic strengths tested. The apparent unwinding angle for chloroquine and 9-amino-1,2,3,4-tetrahydroacridine was found to be ionic strength dependent, increasing with increasing ionic strength. This suggests that competitive nonintercalative binding at low ionic strengths causes an apparent lowering of the quinoline unwinding angle. This can also explain why 4-aminoquinaldine, examined at low ionic strength, gives a quite low apparent unwinding angle. Quinacrine along with chloroquinine and 9-aminoacridine approaches a limiting value for their unwinding angle of approximately 17 degrees. 4-aminoquinaldine and 9-amino-1,2,3,4-tetrahydroacridine could not be examined at an ionic strength above 0.03 because of their very low equilibrium binding constants.     

The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules.

BACKGROUND: The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye-dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy. METHODS: We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO). RESULTS: In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species. CONCLUSIONS: The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission.     

Conformational flexibility of the furanose ring in DNA and its dipole moment

The influence of conformational rearrangement of the furanose ring in DNA on its dipole moment is studied. The dipole moment of the deoxyribose molecule as a function of its puckered state is calculated by the quantum-mechanical method using the MINDO/3 approximation. The values of the dipole moment and its components are obtained at various magnitudes of the pseudorotation phase angle. The C3'-endo in equilibrium C2'-endo conformational transition of deoxyribose is shown to be accompanied by the change in the dipole moment up to 3D. The results obtained are used to explain the structural properties of the DNA hydration shell.     

Physiochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase.

In a medium containing 10mM Tris, pH 8, 10 mM MG++, 50 mM K+ and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 240 degrees at 37 degrees C. In this medium the total unwinding of the DNA increases linearly with the molar ratio of polymerase to DNA. The number of binding sites at which unwinding can occur is very large. If the K+ concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 140 degrees at 37 degrees C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA is strongly temperature dependent, and decreases with decreasing temperature.