Volume 6 Number 10 1979

Nucleic Acids Research

Ethidium bromide binding to core particle: comparison with native chromatin

M.Erard,

G.C.Das*, G.de Murcia, A.Mazen, J.Pouyet, M.Champagne and M.Daune

Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Descartes, 67084 Strasbourg Cedex, France

Received 24 May 1979 ABSTRACT

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.

INTRODUCTION

Ethidium bromide (EB) which was known since a long time as a very convenient probe of DNA structure was used more recently as a tool for determining the structure of DNA inside chromatin (1-15). Two general approaches were made : interpretation of binding isotherms in terms of accessibility and binding constant (1-9) and perturbation of histone-DNA interaction as detected by histone release in presence of both ethidium bromide and NaCl (10-13). We will deal exclusively, in the present work, with the first approach for which two general features have been recognized very early : the existence of two classes of binding sites (1-9) and the presence of clusters of dye molecules (1,7). However miany controversies were present in the literature concerning different aspects of the process : a) the total number of binding sites, b) the presence or not of a plateau in the Scatchard isotherm, c) the interpretation of the first set of binding sites, the so-called high affinity binding sites, d) the role of histone H1. Two explanations can be given for these apparent contradictions : (i) During the last five years considerable progress was made in prepara-

C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

3231

Nucleic Acids Research tion and isolation of chromatin and chromatin sub-units. In the different EB binding studies, samples of chromatin were prepared according to different methods and for example shearing or nuclease digestion did not give comparable material (18). (ii) The methodology was almost exclusively fluorescence measurements, except for few studies using absorbance measurements. Actually a comparison between different physical approaches would be necessary in order to give a more complete picture of the process. In this paper 1) we have carefully prepared and defined the material used and for the

first time experiments are made with core particles. In any case results are averages of many reproducible preparations. 2) Besides fluorescence we have used radioactivity counting with labelled dye , circular dichroism and electron microscopy, and correlation between these different techniques will be carefully discussed. We confirm of course the presence of high affinity binding sites in the chromatin but not in the core particle, i.e. when 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 as far as their binding properties are concerned.

MATERIALS AND METHODS

Preparation of nuclei, chromatin and core particle Nuclei were prepared from mature chicken erythrocytes according to the procedure of Hewish and Burgoyne (16), modified as previously described (17).

Chromatin To the suspension of nuclei was added Micrococcal nuclease (Worthington) (3 units per unit of absorbance at 260 nm) during 30 s at 37°C, releasing X~0.5% acid soluble material. Then lysis of nuclei was achieved by suspending the pellet in 0.2 mM EDTA, pH 7.9. Extracted chromatin was characterized by electron microscopy and circular dichrolsm as described (18). The presence of a superstructure in long chain chromatin as well as the value of the molar ellipticity were among the best evidence of chroma-

tin integrity.

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Nucleic Acids Research

Core_Barticle Nucleosome core particles were prepared according to Lutter (19) except for the last step (manuscript in preparation). Instead of using chromatography in a sepharose 4 B column, the core monomer was separated from nucleotides and residual dinucleosomes by putting the H1 and H5 depleted soluble long chain chromatin redigested with Micrococcal nuclease, on to a 5-30% sucrose gradient in a zonal rotor (38000 rpm during 20 h). Histones of the pure core particles were resolved by electrophoresis on SDS gels according to Laemmli's procedure (20) modified by Weintraub et al. (21). The length of the DNA fragment extracted from the core was measured on polyacrylamide gels according to Peacock and Dingman (22) using Hae III restriction enzyme fragments of PM2 for calibration. DNA DNA (M XV 10 daltons) was extracted from chicken erythrocyte nuclei according to a classical method (23). Measurements

Solutions and buffers were made with analytical grade reagent (MERCK) and bidistilled water. All the experiments were performed in the same medium : 5 mM NaCl, I mM phosphate buffer, pH 7.4, 1 mM EDTA. Ethidium bromide (SERVA, Heidelberg) solution was freshly prepared before each experiment, in this medium. The DNA concentrations of the solutions were determined spectrophotometrically using an extinction coefficient of 6600 at 258 nm. Fluorescence measurements Ethidium bromide (EB) binding to core particles was measured with a SPEX spectrofluorimeter. Excitation and emission wavelengths were 520 nm and 600 nm respectively. The V ratio (7) between fluorescence intensity of bound (IB) and free (IF) dye respectively was first determined. For a given EB concentration (10 M) a complete intercalation in the DNA molecule was obtained with a P/D ratio of 5000. V was found equal to 20.9 in excellent agreement with previous measurements (24). Fluorescence intensity of bound dye at decreasing values of P/D was measured in a single cell, with a constant volume of 3 ml and a total concentration CT of EB maintained equal to 10 M. The concentration P of core particle which initially was 5.7 x -4 10 M (expressed in phosphorus) was decreased stepwise by replacing I ml of the solution by an equal volume of a stock concentration (10 M) of EB.

3233

Nucleic Acids Research The concentration C

of bound EB was measured in each case according to the

classical formula (7) : I

IB

(V The values of A

=

CT

-

CB

- I

F

1)IF

and r = C B/P were then calculated in order to

build the Scatchard plot. Fluorescence polarisation p was determined in the same conditions and the components IVV' IVH' IHV and IHH were respectively measured to get

P

IVV IVV

x

=_ x

IVH IVH

where the ratio x = I HV/IHH was kept constant in our experimental conditions and equal to 1.1.

Eguilibrium dialysis In order to get rid of some difficulties to interpret fluorescence measurements (see results and discussion) EB binding to chromatin and core

particle was followed by equilibrium microdialysis using 14C labelled EB (Modichem Developments Limited). Two cylindrical compartments of 180 pl each, bored in a perspex block were separated by a membrane (Visking). The first one was filled with 150 pl of a solution of 14C EB at concentrations ranging from 1.6 x 10 6M to 3.9 x 10 4 M. The second one was filled with an equal volume of chromatin (core particle) solution at a constant concen-

tration of 1.5 x 10 M. Six plates with four double compartments are clamped together and rotated gently in a thermostated room (23°C) during 24 h, allowing us to obtain in one run twenty values of P/D ratios together with the necessary control experiments. From the specific activity of 14C EB (45 mCi/g), free and bound dye concentrations can be immediately deduced. A long cycle of successive boiling and rinsing of the dialysis membrane, was necessary to avoid EB adsorption to the membrane. Careful decontamination of microsyringes and cells between each run was necessary to reduce the background of counts to a very low value (near the natural radioactivity level in the same conditions of counting). Circular dichro'sm Measurements were made with a Mark III Jobin-Yvon Dichrograph. We used a cylindrical cuvette with 1 cm optical pathlength. Solutions of the 3234

Nucleic Acids Research DNA (or chromatin, or core particles) - ethidium bromide complex for various molar ratios of EB to DNA phosphate (D/P) were prepared by mixing appropriate volumes of the solutions and diluting with buffer to a standard M (P). The Ac = 5L volume. DNA concentration was about 1.5 x 10 values were determined either on the basis of EB (AC308) or on the phosphorus content (Ae275) and expressed (25) in liter mole cm Electron microscoRy

a) The samples used for the CD measurements were fixed in 0.1% glutaraldehyde during one hour at room temperature. They were then diluted to 0.5 pg/ml (expressed in DNA concentration) with 5 mM Triethanol amine, 5 mM NaCl, 0.2 mM EDTA, 0.1% glutaraldehyde, pH 7.4 and spread on positively charged carbon coated grids according to the method of Dubochet (26). Staining and shadowing of the specimen were performed as previously described (18). b) Alternatively, the chromatin - EB or core particle - EB complexes were made at 0.5 pg/ml (expressed in DNA concentration) in a buffer containing 5 mM TEA, 5 mM NaCl, 0.2 mM EDTA, pH 7.4 and the appropriate concentration in EB to obtain the same D/P ratios as in the previous case. After 15 minutes in the dark, the samples were fixed in 0.1% glutaraldehyde during one hour at room temperature and spread under the same conditions as in the previous case. The grids were examined in a Siemens 101 Elmiskop. Calibration was routinely checked by examination of carbon grating replicas (Fullam) photographed after each series of measurements.

RESULTS

1. CHARACTERIZATION OF THE PREPARATIONS

a) Chromatin prepared as indicated, contained long fragments having an average length of about 50 nucleosomes with a range extending roughly from 120 to 8 nucleosomes (see fig. 8a). The protein composition can be seen on the SDS gel shown in fig. Ia. The CD spectrum (see fig. 4a, (1)) of such a preparation is characterized by two well resolved positive peaks at 283 nm and 275 nm and a small negative peak at 296 nm. The maximum molar ellipticity value at 283 nm is 2600 degree.cm 2 .decimole b) Core Rarticle : Gel electrophoresis of the proteins is shown in fig. la. H1 and H5 are completely absent. H2A, H2B, H3 and H4 are found in apparently equal quantities. Gel electrophoresis of the DNA extracted from the par3235

Nucleic Acids Research

Figure 1 - a) Scan of the chromatin and core histones on a b) Scan of the core-DNA on a 6% polyacrylamide SDS-gel. gel stained with ethidium bromide. Sizes of Hae III restriction fragments of PM 2 DNA are taken from Tatchell and Van Holde (27).

ticles is shown in fig. lb. The size of the DNA is 145 ± 3 bp. No evidences for single strand nicks are found in denaturing gels. The particles are homogeneous as seen in fig. 9a. The CD spectrum (see fig. 4b, (1)) of these particles are characterized by a positive peak at 283.5 (101283.5 = 1470 2 degree.cm .decimole -I ), a shoulder at 274 nm and a negative peak at 296 2 (101296 = - 295 degree.cm .decimole -1I ). All the ellipticity values considered

as accurate

within ± 100

degree.cm

2

.decimole

can

be

-

2. BINDING ISOTHERMS In order to check the microdialysis with labelled EB, a preliminary study of EB binding to DNA was made. A linear isotherm, as expected, was obtained (not shown), identical to that obtained from fluorescence measu-

a) DNA

-

rements.

b) Chromatin

-

The binding isotherm of native chromatin (fig. 2) exhibits

a behaviour similar to that obtained when monitoring the process by the fluorescence of intercalated dye molecules. Three binding processes can be

3236

Nucleic Acids Research distinguished : a steep straight line at low r values corresponding to high affinity binding sites already mentioned (1,2,5,7,9), then a plateau value around r = 0.04 - 0.05 (7) followed by a classical exclusion isotherm (28). The total number of binding sites is always difficult to estimate but appears lower than the corresponding value for naked DNA. c) Core particle - In fig. 3 a typical binding isotherm is given, obtained either from counting of labelled dye or from fluorescence. Several striking features are clearly appearing : i) high affinity binding sites are no more present ii) instead a cooperative binding occurs at low r values giving rise to a maximum iii) the slow decrease of r/A0 for r > 0.05 is no more characteristic of the cooperative behaviour but reveals another type of binding as

*

r/A,-io-4

8p

r/A,.10-lo-' Fig. 22

4

4

A

6

g

Fig. 3

0

0

~~~~~~~2

0

2-

o0 0

s05

o;o

aoos005

015

0.10

0.15

0.X0

r

Figure 2 - Scatchard plot obtained by equilibrium dialysis with 14C-ethidium bromide, for nuclease digested chromatin in 5 mM NaCl, 1 mM Phosphate buffer, I mM EDTA (pH 7.4). * Experiment I ; 0 Experiment 2 Figure 3 - Scatchard plot obtained either by fluorescence measurements or equilibrium dialysis with 14C-ethidium bromide, for core particle, in the same buffer as fig. 2. A,A, fluorescence measurements ; D3,- , equilibrium dialysis ; .... Calculated curve (see text). 3237

Nucleic Acids Research discussed further. iiii) the good coincidence between fluorescence and dialysis data in the range 0 - 0.02 of r values, disappears at higher binding ratios. Indeed, even at high D/P ratio the fluorescence intensity does not increase further and therefore the apparent number of intercalated dyes appear to reach a limiting value (fig. 3). It will be shown later (see discussion) that such a behaviour, not found with native chromatin, is easily explained in terms of a strong quenching of EB molecules by energy transfer. In this case any determination of EB binding from fluorescence intensity is largely underestimated. In line with these observations the measurements of polarization fluorescence p (table I) indicate a decrease of p at the immediate beginning of the binding process, pointing to an efficient energy transfer between bound dye molecules and similar to that found with chromatin (7). The extrapolated value of p for r = 0 is smaller than in the case of chromatin since the brownian rotational diffusion constant of the core particle is no more negligible. 3. CIRCULAR DICHROISM From the complete CD spectra recorded at different r values (fig. 4) two wavelengths (308 and 275 nm respectively) were selected for which the corresponding As was plotted against r. The choice of these two wavelengths was dictated by different CD studies on BET - DNA interactions. At 308 nm, the CD signal corresponds to EB - EB interaction and reflects

Table I

r fluo* rdialysis

3238

p

0.0035

0.004

0.326

0.005

0.006

0.311

0.007

0.009

0.296

0.0095

0.014

0.277

0.013

0.021

0.262

0.019

0.031

0.263

Nucleic Acids Research d (in arbilrory units)

d tin arbitrary units) 4

b

a

2160

250

300

320

340 IOU

ibUu

iJ W

s2u A (nm)

X (nm)

Figure 4 - Circular dichrolsm spectra for a) chromatin - BET complexes, b) core particle - BET complexes in the same buffer as fig. 2. 1. r = 0 ; 2. r = 0.01 ; 3. r = 0.03 ; 4. r = 0.10 therefore the vicinity of intercalated dye molecules (25,29). At 275 nm, the CD signal reflects either an induced circular dichrolsm in one of the absorption band of the dye, or a change of the DNA conformation, characteristic of the intercalation. In any case, the corresponding As, when normalized to EB concentration, must be independent of r. If however AC is expressed in nucleotide concentration, the value must be a linear function of r as long as the intercalative process takes place. 308 nm - In the case of core particle (fig. 5), there is an immediate induced CD signal and an almost linear relationship between Ac308 and r, in the range 0 - 0.04. For values of r > 0.04, Ac308 remains constant. The situation is markedly different with DNA and chromatin. In the first case one also observes a linear relationship between Ac308 and r, but the slope is much smaller and the straight line crosses the r axis, not at r = 0, but at r X 0.02. In the case of chromatin, the curve is situated between the two others. The initial linear part begins at r X 0.01, followed by a plateau in the range 0.04 < r < 0.06 which corresponds to that observed in

3239

Nucleic Acids Research the binding isotherms (fig. 2). Apparently the limit value of At308 % 9 is similar for chromatin and core particle, but much smaller than that of about 21 obtained with DNA (30).

275 rm - Since DNA (or chromatin) alone exhibits a positive dichroism at this wavelength we have plotted the difference D275 = Ac r - Acr instead of the absolute value As. The linear variation of D with r found with DNA (fig. 6) in the range 0 < r < 0.1, is also observed with chromatin and core particle but for r < 0.02 with the former and r < 0.04 with the latter. In the case of chromatin a plateau region appears again in the r range 0.04 to 0.06. The limit value at large r values is about the same for both chromatin and core particle but much lower than that observed with DNA. In the three cases, the linear relationship between D and r at the beginning of the process, reflects the local perturbation caused by the intercalation of the dye molecules.

AE 3081

_D275

r Figure 5 - As308 versus r, for DNA (A), chromatin (0 and 0 ), core particle (0). Same conditions as Fig. 2. V value from Aktipis et al. ref. 25 r values were determined from a calibration curve obtained by plotting r as measured in dialysis experiments in function of D/P. Figure 6 - D275 = AEr 0 o - AEr=o versus r for DNA (A), chromatin (0) and core particle (0). Same conditions as fig. 5. r values were determined as in fig. 5. 3240

Nucleic Acids Research 4. ELECTRON MICROSCOPY

a) DNA - As a control of the effect of EB on DNA structure we decided to visualize by electron microscopy the conformational changes of a covalently closed circular DNA due to EB binding. Form I pBR 322 plasmid DNA (kindly provided by Dr M. BACH) in 5 mM Triethanol amine, 100 mM NaCl, 0.2 mM EDTA, pH 7.4 was incubated during 15 minutes in dark at room temperature with various concentrations of EB in the same medium, to obtain the final concentration of 0.5 pg/ml of DNA and a D/P range from 0.01 to 20. The samples were then spread on positively charged carbon coated grids as indicated in the experimental section. The results displayed in fig. 7 show the unfolding

of the twisted tertiary structure leading to its complete relaxation for a value of D/P ratio close to 0.050 (the equivalent point) and ultimately to a refolding in the opposite sense when D/P is equal to 1. When a large excess of EB is added (D/P = 50), the highly twisted four strands induced structure

has a more regular aspect than the starting molecule (D/P = 0). The lengthening of the molecule produced by the intercalation of EB could be checked accurately when pBR 322 form III was used. In agreement with the recent work of Butour et al. (31), we found a 50% length increase for EB saturated pBR 322 form III DNA. Both these qualitative and quantitative results, in full agreement with the hydrodynamic data (32) indicate that in our DNA spreading conditions, there is no measurable interference between the DNA - EB interaction and the EM processing.

b) Chromatin - An aliquot of the chromatin - EB complexes made for the CD experiments were fixed in 0.1% glutaraldehyde and spread as indicated in the experimental section (method a). The results are presented in fig. 8 (a-f). In the untreated chromatin (fig. 8a) nucleosomes in close contact are orga-

nized in a 250 A superstructure fiber (18). At low D/P ratios 0.01 - 0.03 (fig. 8 (b,c)), EB binding produces an unfolding of the superstructure. Nucleosomes are distinct and separated by short DNA like segments. When D/P increases to 0.10 - 0.20 (fig. 8 (d,e)), the length of these segments increases gradually leading to a complete decondensation and linearization of the initial compact structure. At the same time we observe the sliding of certain nucleosomes along the DNA filament, which can explain the creation of dense clusters of histone cores obtained at high D/P values. However the whole population of nucleosomes is not affected by this sliding effect.

3241

a

*'.vi^t0x;.*')XI: > :b

a

Nucleic Acids Research

-

c

~~~~ :.* ... .a¶;t,

* ~

f~~~~~ d t

w . ni $i ;

I

' I+ C~~~~~~, ...0*t'i;t

*

4

vI 494F f..'' ;.*d,'w 5

*4 ~

~

~

~

w

9141

C fC *m y-

I.

-*-

.

..

d

¢ ;>,

5 §e; '0 , t ^*+ t 4 . bi#; xr ^ vb s

4 r =

2

0

o. F............. -.t . . .0. . . ... . .

P T : :''.' 4,~~~~~~~~~~~A.

0

F,2000 A

Figure 8 - Electron microscopy of native chromatin spread according to method a (see experimental section) in the presence of : a) D/P = 0 ; b) D/P = 0.01 c) D/P = 0.03 ; d) D/P = 0.10 ; e) D/P = 0.20 ; f) D/P = 0.30. The Bar indicates 2000 X. 3243

Nucleic Acids Research similarly using both method a and b. The results from method a are displayed in fig. 9 (a-f). In our experimental conditions of staining and shadowing, untreated core particles have an apparent diameter of 140 ± 15 A (fig. 9a). When EB is added in increasing amount a subpopulation of partially unfolded particles appears having the same diameter as the untreated particles but with a DNA tail of variable length. At high D/P ratio (0.20) some free core particle DNA can also be seen (fig. 9f). In contrast to the results obtained by Woodcock and Frado (33) with urea-treated core particles we could detect only one type of morphologically modified core particle, namely a 140 A particle from which extends a DNA tail. We have made an estimation of the proportions of the unfolded particles and the apparently native (without any tail). The propor-

b

d~C

.04

~~'4~~

4~~~~~~ ;

4iX, ' *{4 ;&+;;

,a.

IC :t

0~

~~4

1000 A Figure 9 Visualization of the effect of Ethidium bromide on core particles. Experimental conditions as in figure 8. a) DIP = 0 ; b) D/P = 0.01 ; c) D/P = 0.05 ; d) D/P =00.10 e) D/P = 0.15 ; f) D/P = 0.20 - The bar indicates 1000 A 3244

Nucleic Acids Research tions of free-core particle DNA representing less than 3% of the total population was counted as unfolded particle. The results from two independent experiments (method a and b) are plotted in fig. 10. The initial material contains less than 5% of unfolded particles ; when D/P increases this proportion increases as indicated in the micrographs. A plateau is obtained at D/P % 0.15 - 0.20 for which approximatively half of the population of core particles behave as native, and half as partially or totally (3%) unfolded. This result will be discussed but it is interesting to stress the two points : (i) we have no evidence that the apparently native core particles did not bind EB, (ii) the EB induced DNA unfolding starts from one of the two extremities of the DNA wrapped around the histone core since only one-tail core particles could be observed.

DISCUSSION From the whole set of data, headings for discussion can be

succes-

sively viewed. 1. High affinity binding sites These sites were described in the first studies of EB - chromatin

100%

0 0 X

-,50n-

,.rt

0

0.10

t

0.20

0.30

0.40 D/P

Figure 10 - Variation in percent of the total population of apparently native core particles (closed symbols) and partially or totally denatured core particles due to the binding of EB (open symbols). O core particles without any tail ; 0,A core particles having a DNA tail and yaked core particle DNA - An average of 700 particles were counted for each D/P ratio. 00 (see materials and methods a)) ; AA (see materials and methods b)) 3245

Nucleic Acids Research binding (1,2,4) as correlated with the steep initial part of the Scatchard plot. Unfortunately, at this time, studies were made with sheared chromatin but the high affinity binding sites were also found with nuclease digested chromatin (7,9). In view of the immediate decrease with r of the fluorescence polarization stemming to the presence of intercalated neighbour dye molecules, the high affinity sites were assumed to be situated in the linker DNA (7). This assumption is strenghtened by two of our results : a) in core particle devoided of linker, the high affinity sites are no more present, b) the lengthening and the unfolding of chromatin fiber at low r values is a direct evidence under electron microscope of the linker DNA unfolding and lengthening after completion of the first step of the binding process, i.e. saturation of the high affinity sites. However we will see later that core DNA may also contribute to this structural change and that the two processes occur likely simultaneously. As pointed out by Paoletti et al. (7), this high binding affinity of linker DNA is reminiscent of the behaviour of covalently closed circular DNA towards EB intercalation. The binding constant of the first bound EB molecules is higher than that of linear DNA, because of the contribution of torsional potential energy to the total binding energy (34). Such a behaviour of the chromatin was attributed previously to the presence of H1 (5) since the high affinity sites were no more present after H1 removal. It is now well known that salt removal of H1, if not made very cautiously, is leading to the unfolding of linker DNA (35). However the recovery of the high affinity after H1 reassociation (5) can only be explained in terms of reorganisation triggered by H1 binding and giving rise to a structure of linker DNA similar to that present in the "native" state. 2. Cooperative binding The peculiar shape of the Scatchard plot (fig. 3) is typical of a cooperative binding process. We are thus faced to the problem of interpreting a cooperative binding but also to take into account the exclusion mechanism which is now well documented in the case of intercalating drugs. The best way seems to use the theoretical treatment developed by McGhee and Von Hippel (36) for binding of large ligands to one-dimensional lattices. EB is assumed to cover two base pairs (n = 2 in the terminology of McGhee and Von Hippel) but a cooperativity parameter w is introduced which represents the ratio of the binding constant relative to a dye molecule bound

3246

Nucleic Acids Research close to another one, compared to the binding constant of an isolated dye molecule. By using equation 15 of their paper a set of theoretical isotherms maximum value of r/Ao a are ti hich i computed in functionsof . The initial value of r/Ao close to 6.8 in our experimental isotherm, is used to determine w, since a quasi linear relationship is found between this ratio and w. The best fit appears to be for w = 24 (see fig. 3). In order to have the maximum located at r X 0.04, the maximum number of sites must be equal to 0.06 (expressed per nucleotide). Since the intercept of the isotherm is equal to KN, the intrinsic binding constant is about 2.10 5, where N, the number of potential binding sites, is equal to 0.5 (expressed in nucleotide). Another manner to describe a cooperative process is to assume a conformational change of DNA structure which is reflected in the change of free energy associated to the binding. In other words, the binding of the first dye molecule, by modifying the DNA structure, favored the binding of the next ligand. The simplest manner to describe such a process is to assume AG, the free energy of binding, to be a linear function of r AG

=

AG0 - Ar

where -A is the extra energy per nucleotide provided by the binding of one dye molecule. One gets immediately the equation of the Scatchard's isotherm

r/c = Ko (rm - r) e where rm is the maximum number of sites available in the cooperative process. A plot of Log r/c (r - r) versus r must be linear, with a slope equal to A and an intercept equal to Log K . In order to use this type of plot with our

experimental results we have programmed the minicomputer (C.A.I. LSI2/20) to indicate the best fit when varying both rm and A in a limited range of possible values. With rm = 0.042 and A = 100 ± 5, the line drawn through ten experimental points gives a correlation coefficient of 0.98 and a sum of residues close to 0.29. The corresponding value of Ln K is 12.67 ± 0.09, i.e. a K value of 3.2 ± 0.3 x 10 5 . The fit of this calculated isotherm is similar to that obtained with the first procedure. Without any other data it is difficult to decide between the two interpretations but indeed the results are closed enough to consider the cooperative process as characterized by a maximum number of binding sites per nucleotide in the range 0.04 0.06 and an intrinsic binding constant of the order of 2 - 3 x 10 5 . In any case. several remarks are to be made : a) It is clear after inspection of fig. 3 that the calculated isotherm represents only the initial part of the experimental Scatchard plot. In other 3247

Nucleic Acids Research words, to the cooperative process is superimposed at r values higher than 0.04 - 0.06, another binding process which is represented by the downward part of the isotherm. b) According to the first interpretation, the binding constant of an EB molecule close to another one is 24 times higher than the intrinsic value, i.e. equal to 4..8 106. In the second interpretation when apparent binding constants are calculated in function of r, their values are increasing from 105 (r = 0) to 2.1 107 (r = 0.042) at the end of the process. In 3.2 each case the K value is at least comparable or even higher than that attributed to high affinity binding sites in the case of chromatin. Competition between the two types of sites (high affinity and cooperative) is therefore expected in chromatin. c) The number of cooperative binding sites, relies on the assumption that EB can be equally bound to all of the core particles. This seems indeed to be not the case, since according to electron microscopy, only 50% of the core particles display a protruding DNA of variable length, at the end of the binding process. The other 50% look unmodified. If however, DNA unfolding is assumed to be correlated with cooperative binding (see discussion later), this latter process occurs only into one core particle out of two. Accordingly r values would be two times higher and in the range 0.08 - 0.12. In other words, the core DNA with 290 nucleotides could accommodate cooperatively 24 to 36 EB molecules per "receptive" core particles, which are located in a segment of 48 to 72 base pairs respectively. It is now possible to compare the cooperative binding in core particle with that occuring in chromatin. It is clear that the plateau value in the binding isotherm of chromatin (fig. 2) corresponds to the superimposition of an exclusion isotherm relative to the linker (high affinity binding sites) and a cooperative binding isotherm relative to the core particle. According to remark b) this latter process becomes rapidly competitive and then dominant compared to the first one. .

.

.

m

When isotherms are obtained with sheared chromatin (5,7), this plateau-shaped isotherm is no more found. This could be an indication that during the preparation a partial unfolding of the core DNA has occured which affects that part of the DNA responsible of the cooperative binding process. Such a structural perturbation at the nucleosome level was already pointed out (18). The comparison between chromatin and core particle relies on an implicit assumption : the structure of the core particle is the same when inside the chromatin or isolated. It does not mean however, as it will be 3248

Nucleic Acids Research discussed later, that the trigger of the cooperative process is the same in the two materials. 3. Clustering

of EB leads to a cluster of dye molecules, the existence of which is confirmed by circular dichroism and fluorescence polarisation. a) At 308 nm, the CD signal is considered as induced by a coupling between the transition dipoles of neighbour EB molecules (25,29). As soon as the first EB molecules are bound to core-DNA, Ac308 is measurable and increases linearly with r up to r 'v 0.04 - 0.06, i.e. when the cooperative binding sites are saturated. In the case of naked DNA, a CD signal occurs at 308 nm but only for r > 0.02 and its increase with r is much slower. It reflects the random appearance of dye-pairs but without any systematic cooperative clustering. The biphasic shape of Ac308 (r) in the case of chromatin is characteristic of at least a two-step process. The first part presents an initial slope similar to that observed with the core particle but AC308 is measurable only when r > 0.01. Such a behaviour is typical of a competition initially present in chromatin between high affinity binding sites in the linker and cooperative sites in core DNA. The anticooperative mode of binding in the linker is not favorable to the formation of dye pairs which explains the absence of CD signal in the range 0 < r < 0.01. The plateau which occurs for r "X 0.04 in the case of chromatin corresponds to Ac308 X 6 that is 2/3 of the limiting value of 9 obtained with core particle. In other words, when r 'v 0.04 about 2/3 of bound EB molecules are clustered in the core DNA, since Ac308 values are expressed in terms of the concentration of bound EB. b) Interpretation of fluorescence polarisation p is slightly different since in this measurement energy transfer can take place at distances higher than that of 10 A which corresponds to the closest distance of two EB molecules. The cooperative binding

Clustering, as measured by the fast decrease of p with r, is thus different from that detected by the value of Ac308' The second one, when present, gives rise also to depolarisation of fluorescence but the reverse is not true. Careful calculation, using precise relative geometry of the bound dyes would be necessary to determine the extent of the cluster as observed in both cases: high affinity sites and cooperative binding. So far we can say that the measurement of Ac308 is the only physical probe able to distinguish between the two types of clusters.

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Nucleic Acids Research 4. Non cooperative binding After the formation of EB cluster in the core particle or after the filling of both the high affinity sites and the cooperative sites in chromatin, another binding process takes place leading to a final value of r X 0.17 for chromatin but close to 0.25 for core particle. From inspection of the results it is clear that this binding process cannot be the same for chromatin and core particle, and we have to discuss separately their respective behaviour. a) Chromatin - Since the isotherm determined from dialysis experiments with labelled dye is superimposed to that deduced from fluorescence measurement, the binding process which occurs at r > 0.06 is obviously an intercalation. An evidence for intercalation is also provided by Ac308 values, which in the same r range are again increasing with r (fig. 5) in a manner similar to that found with DNA. The apparent limit r value of about 0.17 (fig. 2) could be misleading, in view of the curvature of any exclusion isotherm at the end. However the limit value of Ac308 is less than half that measured with DNA alone, leading to a qualitative conclusion about a partially masked DNA inside chromatin. Opposite conclusions, which were obtained previously after a careful analysis of the isotherms (5) could likely be explained in terms of partially sheared and then more accessible chromatin. b) Core particle - The picture is drastically different because of the apparent limit value close to 0.25 (fig. 3) and because of the absence of any extra CD signal at 308 nm when r > 0.06 (fig. 5). Furthermore, the downward part of the isotherm was only obtained from dialysis measurement and this technique does not allow to distinguish between intercalative and outside binding. We think therefore that upon completion of the cooperative filling of a part of the core DNA, EB molecules can only be bound via coulombic interactions, especially in a low ionic strength me-

dium. 5. Structural changes and heterogeneity of core particles It is clear from the pictures (Figs 8,9) that EB binding triggers the unfolding of DNA inside chromatin or core particle. When r reaches its limit value (around 0.2) the chromatin structure is dramatically altered and the reversibility of such a structural change is now beeing investigated in our laboratory. The CD signal at 275 nm could be considered as a direct indication of DNA structural changes, but the contribution of an induced CD signal in

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Nucleic Acids Research the absorption band of the dye cannot be excluded. The two sets of points corresponding to DNA and core particle respectively are situated in the same straight line, in the range 0 < r < 0.05, which indicates a conformational change of the core DNA during the cooperative binding similar to that of naked DNA. If now a correlation is made between the CD data and EM microscopy which shows the presence of DNA "tails" at only one end of the core, a tentative explanation for cooperativity could be proposed. The binding of EB to one end of core DNA induces a stripping of the DNA helix from the core histones. As it was previously pointed out (37), the effective ionic strength experienced by the DNA inside the core particle, is closed to 0.1 M salt. As soon as the first EB molecules are bound, the DNA chain stretches out progressively in a medium of low ionic strength and the electrostatic part of the binding energy is increasing strongly, giving rise to the observed increase of the binding constant. It is clear, in this case, that the change of D275 with r is similar to that observed with naked DNA. Finally we have to find some kind of explanation for the heterogeneity of core particles. A figure of 50% points to some random process taking place during digestion by Micrococcal nuclease and giving rise to two different types of interaction between the end of DNA chain and the core histones. In only one case could the DNA be stretched out under the unwinding constraint induced by EB intercalation. Careful examination of the digestion pattern would be necessary as well as the isolation of each type of core particle.

CONCLUSIONS

1) The high affinity binding sites are situated in the linker and HI in agreement with unpublished results on H1 depleted chromatin in which high affinity binding sites are still pre-

not correlated with the presence of sent.

2) The cooperative binding occurring in core particle could be

explained in terms of a progressive stripping and unfolding of the DNA out of the core histone. A similar process must take place in chromatin. 3) This specific sensitivity of the end region of the core DNA, is only observed with one core particle out of two. These two subclasses are likely related to some random character of enzymatic cleavage of the phosphodiester bond. In the line of these three new aspects of EB binding to core particle and chromatin, studies are now in progress in our laboratory

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Nucleic Acids Research ACKNOWLEDGEMENTS The technical assistance of J. DUNAND and D. BUHR was highly appreciated. We are indebted to Y. MAUSS and Pr J. CHAMBRON for kindly providing their fluorescence equipment. We must thank J. PAOLETTI, D. DOENECKE, J.J. LAWRENCE and H. GENEST for fruitful and stimulating discussions.

One of the authors (G.C.D.) is indebted to the French Ministry of Foreign Affairs for offering him a post-doctoral fellowship. This work was supported in part by a grant of the CNRS (ATP Chroma-

tine, contract n° 2869).

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