Fluorescence anisotropy decay of ethidium bound to chromatin

The fluorescence anisotropy decays of the chromatin ethidium complexes have been measured in solutions in which the dye was bound to the high affinity sites of the nucleosome DNA. Energy transfers between chromatin-bound ethidium molecules cause an increase of the anisotropy decay rate for much smaller values of the concentration ratio of dye to nucleotide than in the case of nacked DNA-ethidium complexes. This result implies that the high affinity sites are clustered on a short nucleosomal DNA segment. Quantitative analysis of the experimental data by computer simulations of the energy transfer process, shows that these sites are gathered on a single nucleosomal DNA segment, 28 base pairs long. Such a segment probably belongs to the nucleosome “linker”, contributing about half of it.     

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.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

Kinetics of nucleosome unfolding at low ionic strength

The kinetics of a conformational change which occurs in nucleosome core particles at about 1 mM ionic strength have been studied by observing changes in the fluorescence of labeled histone H3. The unfolding reaction is intramolecular since no concentration dependence is observed. However, the kinetics are unexpectedly complicated and reveal evidence of at least three relaxation times. It is possible to fit the kinetics observed under several conditions to a consistent four-state cyclic mechanism in which folded and unfolded forms can inter-convert by two parallel pathways, each involving a distinct intermediate. While the data are not sufficient to establish this mechanism as a unique choice, they exclude many simpler possibilities. The cyclic mechanism is quite reasonable in view of what is currently known about the structures of the folded and unfolded forms.     

Neutron-scattering studies of accurately reconstituted nucleosome core particles and the effect of ionic strength on core particle structure

Chicken erythrocyte nucleosome core particles can be dissociated quantitatively into histones (H3, H4)2 bound to 146 base pairs of DNA, and 2(H2A, H2B). Reconstitution of core particles from the two components produces an 85% yield of particles which neutron scattering studies show to be accurate stoichiometrically and indistinguishable from native core particles: the radii of gyration of the shape, the protein components and the DNA components of the particles are 4.02 nm, 3.3 nm and 4.95 nm respectively. The largest distance and most probable distance which can be drawn in the particles are 11.5 nm and 4.3 nm respectively. The molecular weight of the particles is identical to that of control 'native' core particles. All of these values, within limits of error, are the same as known values for 'native' core particles. These experiments confirm the essential role of histones H3 and H4 in the initial organisation of core-particle structure, make possible the manufacture of perfectly pure and homogeneous core-particle preparations and allow the 100% incorporation of labelled or modified histones. Neutron scattering studies of core particles at high contrast (in D2O and H2O) have been carried out over a range of ionic strengths and pH. No change in structure is detected down to pH 5.5 in 20 mM NaCl or down to ionic strength 2.0 mM at pH 7.     

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.     

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.     

Theoretical study of structural transition in a nucleosome at low ionic strength

The theoretical analysis of nucleosome stability at low ionic strength has been performed on the basis of consideration of different contributions to the free energy of compact state of the nucleosome DNA terminal regions. The proposed model explains: the fact of low-salt structural change; the transition point (approximately 1.7 mM NaCl) and width (approximately 1 mM); the shift of the transition to the higher salt concentrations in the case of histones tails removal by trypsin. According to the model the increase of electrostatic repulsion between neighbouring turns of DNA superhelix is the main cause of the unwinding of nucleosomal DNA terminal regions in the course of low-salt structural change. The interactions between histone (H2A-H2B) dimer and (H3-H4)2 tetramer provide the compact state of the nucleosomal DNA terminal regions. The existence of electrostatic interactions of nucleosomal DNA terminal regions with tetramer was suggested. These interactions can provide the compact state of nucleosomal DNA at physiological ionic strength even in the absence of (H2A-H2B) dimer.     

Fluorescence anisotropy decay due to rotational brownian motion of ethidium intercalated in double strand DNA

The transient fluorescence of solutions of ethidium bromide . DNA complexes has been measured by pulse fluorimetry at different temperatures and in solvents containing various amounts of sucrose. The molar ratio of ethidium to nucleotides was low. Under these conditions the anisotropy decay was due to the Brownian motion of ethidium molecules intercalated in the double strand DNA molecules. This anisotropy decay could be described by a sum of 3 exponential terms, with correlation times 01, 02, 03 which were linear functions of the ratio of the solvent viscosity to the absolute temperature (n/T). The amplitude of the exponential term characterized by the shortest correlation time (01) has been found to depend on temperature while the ratio of the amplitude of the two other terms (characterized by 02 and 03) was independent of temperature. These results were interpreted as follows: 01 corresponds to a fast motion of the dye in its site. 02 and 03 describe a tortional motion of the ethidium bromide. DNA complex, involving several nucleotide pairs.     

The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.     

Binding of ethidium to the nucleosome core particle. 2. Internal and external binding modes

We have previously reported that the binding of ethidium bromide to the nucleosome core particle results in a stepwise dissociation of the structure which involves the initial release of one copy each of H2A and H2B (McMurray & van Holde, 1986). In this report, we have examined the absorbance and fluorescence properties of intercalated and outside bound forms of ethidium bromide. From these properties, we have measured the extent of external, electrostatic binding of the dye versus internal, intercalation binding to the core particle, free from contribution by linker DNA. We have established that dissociation is induced by the intercalation mode of binding to DNA within the core particle DNA, and not by binding to the histones or by nonintercalative binding to DNA. The covalent binding of [3H]-8-azidoethidium to the core particle clearly shows that less than 1.0 adduct is formed per histone octamer over a wide range of input ratios. Simultaneously, analyses of steady-state fluorescence enhancement and fluorescence lifetime data from bound ethidium complexes demonstrate extensive intercalation binding. Combined analyses from steady-state fluorescence intensity with equilibrium dialysis or fluorescence lifetime data revealed that dissociation began when approximately 14 ethidium molecules are bound by intercalation to each core particle and less than 1.0 nonintercalated ion pair was formed per core particle.     

Evidence for an increase of DNA contour length at low ionic strength.

The polyion chain expansion of DNA was studied by viscometry within the Na+ concentration range c5 = 0.002 M to 0.4 M. The DNA molecular weights M were between 0.5 x 10(6) and 13 x 10(6). The relative change of intrinsic viscosity [eta] is linearly correlated to c5(-1/2) with a slope that increases with increasing M. This behaviour reflects the predominance of helix stiffening in chain expansion. At c5(112) > 0.01(-1/2 M-1/2 (Debye-Hückel screening radius 1/chi > (1/chi)*=3nm) the relative change of [eta] rises with a steeper slope. This effect increases with decreasing M suggesting that helix lengthening contributes to the chain expansion. Our model enables us to interpret other ionic-strength dependent effects known from literature. The start of the significant duplex elongation at (1/chi)* can be correlated to the polyion-charge arrangement. In accordance with our interpretation (1/chi)* is found to be greater for DNA-intercalator complexes.     

Analysis of the changes in the structure and hydration of the nucleosome core particle at moderate ionic strengths

In order to better understand the conformational changes induced in the nucleosome core particle by changes in the ionic strength of the media in the range from 0.1 to 0.6 M NaCl, we have conducted a very detailed structural analysis, combining circular dichroism, DNase I digestion, and sedimentation equilibrium. The results of such analysis indicate that the secondary structure of both DNA and histones exhibits small (approximately 5%) but noticeable changes as the salt increases within this range. In the case of DNA, the data are consistent with a trend toward a more relaxed secondary structure. The DNase I pattern of digestion is also altered by the salt and suggests a DNA relaxation around the flanking ends. From the hydrodynamic measurements, we also observe a significant change in the virial coefficients of the particle as the salt increases, which in turn are in very good agreement with the theoretically expected values. Furthermore, the preferential hydration parameter is also found to increase with the salt. We believe that the self-dependent conformational change of the nucleosome core particle is the result of the conjunction of all these subtle changes. Yet, from the present data, their exact relationship to the tertiary structure of the whole particle at the different ionic strengths cannot be exactly defined.     

The roles of H1, the histone core and DNA length in the unfolding of nucleosomes at low ionic strength.

Calf thymus nucleosomes exhibit two different and independent hydrodynamic responses to diminishing salt concentration. One change is gradual over the range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-site cross-linking efficiency. The other change is abrupt, being centered between 1 and 2 mM Na+. We found only one abrupt change in sedimentation rate for particles ranging in DNA content fom 144 to 230 base pairs. This response to decreasing ionic strength is similar for particles of both 169 and 230 base pairs. Core particles (144 base pairs) exhibit a somewhat diminished response. The abrupt change is blocked by formaldehyde or dimethylsuberimidate cross-linking. The blockage by dimethylsuberimidate demonstrates that the abrupt conformational change requires the participation of the core histones. H1 completely blocks the abrupt but not the gradual conformational change. Thus H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.     

1H NMR investigation of the conformational states of DNA in nucleosome core particles.

In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec.     

A comparison by ultracentrifugation of the effects on DNA of ethidium bromide and of acridine orange at low ionic strength

The application of scaled particle theory to the gels formed by DNA in the ultracentrifuge has provided values for the effective length and the effective radius of the DNA particle. Ethidium bromide has been shown to cause extensive lengthening of the DNA in dilute salt. Acridine orange interaction with DNA resulted in modest changes in DNA dimensions. These results are explained in terms of binding for acridine orange and of denaturation of DNA by ethidium bromide.     

Selective removal of histone H1 from nucleosomes at low ionic strength

The method for removal of histone H 1 from chromatin by treatment with ion-exchange resin AG 50 WX 2 in the presence of 100 mM NaCl and 50 mM phosphate buffer (Thoma and Koller, 1977, Cell, 12, 101–107) results in production not only of H1-depleted chromatin but also free DNA. We have now modified this procedure so that the nucleosome is treated with the cation exchange resin in two steps, first in 50 mM sodium phosphate buffer and then in 50 mM sodium phosphate and 50 mM NaCl whereby histone H 1 is selectively removed without a release of free DNA at low resin concentrations.Abbreviations NaP Sodium phosphate buffer of molarities and pH as stated in the text - SDS Sodium dodecyl sulfate     

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.