AFM Imaging of SWI/SNF action: mapping the nucleosome remodeling and
  sliding
Montel_2007_condmat
AFM imaging of SWI/SNF action : mapping the 
nucleosome remodeling and sliding 
Fabien MONTEL, Emeline FONTAINE, Philippe ST-JEAN, Martin CASTELNOVO 
and Cendrine FAIVRE-MOSKALENKO 
Laboratoire Joliot-Curie (CNRS USR 3010) et Laboratoire de Physique (CNRS UMR 5672), 
Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69007 Lyon, France 
ABSTRACT 
We propose a combined experimental (Atomic Force Microscopy) and theoretical study of the 
structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, 
this method allows to determine simultaneously the DNA complexed length distribution and 
nucleosome position in various contexts. First, we show that differences in the nucleo-proteic 
structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce 
quantitative changes in the in the length distribution of DNA complexed with histones. Then, 
the sliding action of remodeling complex SWI/SNF is characterized through the evolution of 
the nucleosome position and wrapped DNA length mapping. Using a linear energetic model 
for the distribution of DNA complexed length, we extract the net wrapping energy of DNA 
onto the histone octamer, and compare it to previous studies.  
Keywords : Atomic Force Microscopy, mono-nucleosome, H2A.Bbd, length distribution of 
wrapped DNA, nucleosome position distribution, chromatin remodeling factor, histone 
variant 
INTRODUCTION 
DNA is packaged into chromatin in the cell nucleus. The chromatin repeating unit, 
called the nucleosome, consists of an octamer of the core histones (two each of H2A, H2B, 
H3 and H4) around which about two superhelical turns of DNA are wrapped (1). The 
Nucleosome Core Particle (NCP) represents a barrier for the transcription factors binding to 
their target DNA sequences and interferes with several basic cellular processes (2). Histone 
modifications, ATP-remodeling machines and the incorporation of histone variants within 
chromatin are used by the cell to overcome the nucleosomal barrier and modulate DNA 
accessibility by the control of nucleosome dynamics (3-6). In this work, we use a single 
molecule technique (Atomic Force Microscopy) to visualize isolated mono-nucleosomes, to 
quantify the influence of histone octamer composition (H2A-Bbd variant) on the equilibrium 
nucleosome conformation and to map nucleosome mobility induced by a remodeling complex 
(SWI/SNF). 
Chromatin remodeling complexes are used by the cell to overcome the general 
repression of transcription associated with the DNA organization into chromatin (7-9). In 
order to destabilize histone-DNA interaction, remodeling factors (like SWI/SNF) consume the 
energy from ATP hydrolysis to relocate the histone octamer along the DNA sequence (10, 11) 
and in some cases, the ejection of the octamer from the DNA template is observed (12). The 
molecular motor SWI/SNF is known to mobilize the histone octamer from a central to an end-
position on short DNA templates (13). Nevertheless, the molecular mechanisms involved in 
the nucleosome remodeling process have not yet been elucidated .  
Histone variants are nonallelic isoforms of the conventional histones. The function of the 
different histone variants is far from clear, but the emerging general picture suggests that the 
incorporation of histone variants (14-19) in the nucleosome has serious impacts on several 
processes, including transcription and repair, and it may have important epigenetic 
consequences (20-23). H2A.Bbd (Barr body deficient) is an unusual histone variant whose 
primary sequence shows only 48% identity compared to its conventional H2A counterpart 
(24). The current view is that H2A.Bbd is enriched in nucleosomes associated with 
transcriptionally active regions of the genome (24). In recent studies, the unusual properties of 
this variant nucleosome were described (16, 25) using a combination of physical methods and 
molecular biology approaches. Those results were mainly focused on the biological role of the 
various histone fold domains of H2A.Bbd on the overall structure, stability and dynamics of 
the nucleosome, whereas we concentrate here on the quantification of the subtle modifications 
in the nucleosome conformation induced by the presence of this histone variant.  
Different experimental approaches have been used so far to study the structure and 
dynamics of the nucleosome, including crystallographic studies by Luger et al. (26) , 
restriction enzyme accessibility assays (27, 28), and FRET measurements (29, 30). 
Additionally, physical models (31) and recent computational efforts were developed to 
describe the nucleosome dynamics and energetics (32-35). Following these numerous 
contributions, the present study combines experimental (Atomic Force Microscopy) and 
theoretical tools to bring complementary information regarding the interplay between 
nucleosome position dynamics and DNA wrapping energetics. 
Atomic Force Microscopy (36) allows direct visualization of chromatin fibers and 
isolated nucleosomes (37). Several experimental procedures allow to depose and observe 
reproducibly, all this without any fixing agent, DNA or chromatin samples (38-44). By 
scanning the sample with an apex of very high aspect ratio mounted on a flexible lever, the 
topography of a surface at the nanometric scale can be acquired. Moreover, computer analysis 
of AFM images enables the extraction of systematic and statistically relevant distributions of 
structural parameters describing these biological objects (45-47). As nucleosome is a complex 
and very dynamic structure, it has been observed that, for a given DNA template, the position 
of the octamer relative to the sequence (13, 48-50) and the length of DNA wrapped around the 
histone octamer (27-29, 51, 52) both could change drastically in time.  
This paper is organized as follows. First, we show that mapping the nucleosome 
position along with the length of DNA complexed with histones within individual nucleosome 
is a powerful tool to discriminate between conventional and variant nucleosomes. A model is 
then proposed to explain quantitatively these differences and to calculate the wrapping energy 
of nucleosomes in each case. Next, we have studied nucleosomes in a more dynamic context 
by observing the action of chromatin remodeling factor SWI/SNF. To do so, similar mapping 
of the nucleosome position and DNA complexed length was used to quantify the impact of 
ATP-activated remodeling and sliding of nucleosomes. The results suggest experimental 
insights into the processivity of SWI/SNF on mono-nucleosomes. 
MATERIALS AND METHODS  
Preparation of DNA fragments 
The 255 bp and 356 bp DNA fragments, containing the 601 nucleosome positioning 
sequence(53), were obtained by PCR amplification from plasmid pGem-3Z-601. For the 
255 bp template, 147 bp long 601 positioning sequence is flanked by 52 bp on one side and 
56 bp on the other side. For the 356 bp template, 147 bp long-601 positioning sequence is 
flanked by 127 bp on one side and 82 bp on the other side. As both 601 DNA templates are 
built from the same plasmid, the DNA flanking sequences of the short template are included 
in the long DNA template. 
Protein purification, nucleosome reconstitution and remodeling  
Recombinant Xenopus laevis full-length histone proteins were produced in bacteria 
and purified as described (54). For the H2A.Bbd protein, the coding sequences for the H2A 
and for H2A.Bbd were amplified by PCR and introduced in the pET3a vector. Recombinant 
proteins were purified as previously described (55). 
Yeast SWI/SNF complex was purified as described previously (56) and its activity 
was normalized by measuring its effect on the sliding of conventional nucleosomes : 1 unit 
being defined as the amount of ySWI/SNF required to mobilize 50% of input nucleosomes 
(~50 ng) at 29°C during 45 minutes. Nucleosome reconstitution was performed by the salt 
dialysis procedure (57). Nucleosomes reconstituted on a 601 nucleosome positioning 
sequence (20 ng) were incubated with SWI/SNF as indicated at 29°C and in remodeling 
buffer (RB) containing 10 mM Tris-HCl, pH = 7.4, 2.5 mM MgCl2, and 1 mM ATP. The 
reaction was stopped after the time as indicated by diluting about 10 times in TE buffer (Tris-
HCl 10 mM, pH = 7.4, EDTA 1 mM) and NaCl 2 mM and deposing the sample onto the 
functionalized APTES-mica surface. 
Atomic Force Microscopy and surface preparation 
For the AFM imaging the conventional and variant nucleosomes were immobilized 
onto APTES-mica surfaces. The functionalization of freshly cleaved mica disks (muscovite 
mica, grade V-1, SPI) was obtained by self-assembly of a monolayer of APTES under Argon 
atmosphere for 2 hours (39). Nucleosomes (DNA concentration ~ 75 ng/µl) were filtered and 
concentrated using Microcon® centrifugal filters to remove free histones from the solution, 
and diluted 10 times in TE buffer, just prior to deposition onto APTES-Mica surfaces. A 5 µl 
droplet of the nucleosome solution is applied on the surface for 1 min, rinsed with 1 mL of 
milliQ-Ultrapure © water and gently dried by nitrogen flow. The samples were visualized by 
using a Nanoscope III AFM (Digital Instruments™, Veeco, Santa Barbara, CA). The images 
were obtained in Tapping Mode in air, with silicon tips (resonant frequency 250-350 kHz) or 
Diamond Like Carbon Spikes tips (resonant frequency ~150 kHz) at scanning rates of 2 Hz 
over scan areas of 1 µm wide.  
This surface functionalization was chosen because it is known to trap 3D conformation 
of naked DNA molecule on a 2D surface (58, 59). Moreover, under such experimental 
conditions, rinsing and drying are thought to have little effect on the observed conformation 
of biomolecule (60).  
Image analysis 
We have extracted parameters of interest from the AFM images using a MATLAB© 
(The Mathworks, Natick, MA) script essentially based on morphological tools such as binary 
dilatation and erosion (61-64), and height/areas selections . The aim of the first three steps of 
this algorithm is to select relevant objects : 
1. In order to remove the piezoelectric scanner thermal drift, flatten of the image is 
performed. The use of a height criteria (h>0.5nm where h is the height of the object) 
allows to avoid the shadow artifact induced by high objects on the image. 
2. Building of a binary image using a simple thresholding (h > 0.25 nm where h is the 
height of the object)) and then selection of the binary objects in the good area range 
(500 < A < 2000 nm² where A is the area of the object)). 
3. Selection of the objects in the good height range using a hysteresis thresholding (65) 
(hmin1 = 0.25 nm and h
2 = 1.4 nm, where h
1 and h
2 are the height of the two 
thresholds).   
These three steps leads to the selection of binary objects whose area is between 500 
and 2000 nm² and corresponds in the AFM image to a group of connected pixels whose 
minimun height is more than 0.25 nm and maximum height is above 1.6 nm. For example a 
height criterion is used to reject tetrasomes while events with SWI/SNF still complexed with 
nucleosomes are removed from analysis by a size criterion. The next steps correspond to 
measurements in itself : 
4. Detection of the NCP centroid by shrinking the objects in the binary image. 
5. Building of a distance map inside the nucleosome with respect to their NCP centroid 
using a pseudo-euclidian dilatation based algorithm. 
6. Selection of the non-octamer parts of the nucleosomes (d > dc , where d is the 
constraint distance to the NCP centroid and dc ~ 7.5 nm is the apparent nucleosome 
radius) and then thinning of the free arm regions using a commercial MATLAB© 
script optimised to avoid most of the branching in the skeleton. 
7. Selection of the free arm ends and measurement of the free arm lengths.  
8. Measurement of other parameters of interest like areas, volumes and mean height of 
the nucleosomes and the octamers (see supplemental materials). 
These last 5 steps lead to quick and robust measurements. Indeed the use of morphological 
tools allows  parallel calculation simultaneously on all the objects. Moreover, erosion is a 
good approximation for the inverse operation of the AFM dilatation due to the finite tip radius 
and leads to a partial removal of the tip effect (66, 67). 
The longest arm is named L+ and the shortest L-. DNA complexed length is deduced 
by Lc = Ltot - L- - L+ where Ltot is either 255 bp for short conventional and variant 
nucleosomes or 356 bp for long conventional nucleosomes. The position of the nucleosome 
relatively to the DNA template center is calculated as ∆L = (L+ - L-)/2. Notice that the 
position defined this way corresponds to the location of the most deeply buried base pair, 
which might differ from dyad axis position (strictly defined for symmetric nucleosomes). 
Complexed DNA length and nucleosome position distribution construction 
For the distribution of DNA complexed length, well centered nucleosomes were 
selected (∆L* - σ∆L/2 ~ 0 bp < ∆L < 12 bp ~ ∆L
* + σ∆L/2 for the 255 bp mono-nucleosomes 
where ∆L* is the most probable nucleosome position and σ∆L is the standard deviation of the 
∆L distribution). To construct the histogram a 20 bp-sliding box was used. For each L0 in [0, 
300 bp], nucleosomes with a DNA complexed length included in the range [L0 – 10 bp, 
L0 + 10 bp] were counted. After normalization, a smooth distribution is obtained that 
represents mathematically the convolution of the real experimental distribution with a 
rectangular pulse of 20 bp long. 
To obtain the nucleosome position distribution we have selected nucleosomes with a 
DNA complexed length Lc in a range of width σLc around L
* = 146bp 
(123 bp ~ L* - σLc < Lc < L
* - σLc 169 bp for canonical nucleosomes). Then, the same 20-bp 
sliding box protocol was used to construct the nucleosome position distribution. The error on 
the distribution function mean value (standard error) is given by σexp/√N, where σexp is the 
standard deviation of the experimental distribution, and N the number of analyzed 
nucleosomes (central limit theorem). 
2D distribution Lc/∆∆∆∆L construction 
To construct the 2D-histogram a 10 bp-sliding box was used. For each coordinates 
(∆L0, L0) in [0, 75 bp]×[0, 300 bp], nucleosomes with a DNA complexed length included in 
the range [L0 – 5 bp, L0 + 5 bp] and a position included in the range [∆L0 – 5bp, ∆L0 + 5 bp] 
were counted. After normalization a smooth distribution is obtained that represents 
mathematically the convolution of the real experimental 2D-distribution with a 10 bp square 
rectangular pulse. 
Reproducibility and experimental errors 
We have checked that different batches of APTES, nucleosome reconstitutions, 
ySWI/SNF and mica surfaces lead to similar results for the sliding assays and for the 2D 
mapping within the experimental uncertainty. Moreover we have checked by image analysis 
of the same naked DNA on the same surface and within the same experimental conditions 
(data not shown) that the whole measurement and analysis process have an experimental error 
of about 10 bp in DNA length measurement. Notice that uncertainty on the mean value of 
length measurements can be much smaller than this resolution as it is explained in the 
supplemental material S3. 
RESULTS AND DISCUSSION 
Simultaneous measurements of DNA complexed length and 
nucleosome position. 
Several biochemical approaches allow accessing either the nucleosome position along 
a DNA template, or the length of DNA wrapped around the histone octamer, but using AFM, 
we were able to measure them simultaneously. The results are conveniently plotted as 2D 
histograms of nucleosome position versus DNA complexed length. 
For short and long arm mononucleosomes  
We first investigated the influence of the DNA template length on the nucleosome 
complexed length distribution for conventional nucleosomes. Indeed, one could expect that 
the nucleosome positioning efficiency for the 601 DNA template and/or the range of wrapped 
DNA length could depend on the length of free DNA arms. Using purified conventional 
recombinant histones, nucleosomes were reconstituted by salt dialysis on 255 bp (short 
nucleosomes) or 356 bp (long nucleosomes) DNA fragments containing the 601 positioning 
sequence. Tapping Mode AFM in air was used to visualize the reconstituted particles 
adsorbed on APTES-mica surfaces and images of 1 µm2 were recorded. A representative 
image of long mono-nucleosomes (Ltot = 356 bp) is displayed on Figure 1a. Such an image 
enables to clearly distinguish the nucleosome core particle (red part of the complex : hNCP ~ 2 
nm) from the free DNA arms (yellow part of the complex, hDNA ~ 0.7 nm) entering and 
exiting the complex.  
Precise measurement of the length of each DNA fragment (respectively L+ and L- for 
the longer and shorter arm) exiting the nucleosome have been performed. To measure each 
“arm” of the mono-nucleosome, the octamer part is excluded and the free DNA trajectory is 
obtained (Fig.1b) using morphological tools avoiding false skeletonization by heuristic 
algorithm ( cf Material and Methods). From the total DNA length that is un-wrapped around 
the histone octamer, we get the length of DNA organized by the histone octamer 
(Lc = Ltot - L+ - L-) as well as the nucleosome position with respect to the center of the 
sequence (∆L = (L+ - L-)/2). 
The 2D histogram Lc/ ∆L is plotted on Fig. 1c for 702 conventional short nucleosomes 
using a 2D sliding box as described in the Material and Methods section. The maximum of 
the 2D distribution is positioned at L* = 145 bp and ∆L = 15 bp, in qualitative agreement with 
the DNA template construction. The 2D mapping is an important tool to study nucleosome 
mobilization (see the SWI/SNF sliding section), since both variables are highly correlated 
during nucleosome sliding/remodeling. Quantitative information can be however also 
obtained by projecting such a 2D histogram on each axis. First, we have selected well 
positioned nucleosomes according to the expected position given by the DNA 601 template 
construction (0 bp < ∆L < 12 bp for short DNA fragments) and shown their DNA complexed 
length probability density function (red line, Fig. 1d). This distribution of the DNA length, 
organized by conventional octamer peaks at L* = 146 ± 2 bp, in quantitative agreement with 
the crystal structure of the nucleosome (26) and cryoEM measurements (25). The broadness 
of this distribution (σ = 23bp) might be explained by different nucleosomes wrapping 
conformations. We will explain later on, how this dispersion relates to DNA-histone 
interaction energies using a simple model. 
We have used the same approach to study long nucleosomes (2D histogram not 
shown). Well positioned long nucleosomes according to the DNA sequence 
(12 bp < ∆L < 32 bp) have very similar probability distribution (blue line on Fig. 1d) than that 
obtained for short nucleosomes showing that the free linker DNA does not affect significantly 
the organization of complexed DNA for such nucleosomes.  
We now select nucleosomes that have a complexed length in the range L* ± σLc, where 
σLc is the standard deviation of the Lc distribution, and their position distribution is displayed 
on Figure 1e. The peak values for each DNA fragment (9 ± 2 bp and 24 ± 2 bp for short and 
long nucleosomes respectively) is close to the expected value from the DNA template 
construct (2 bp and 22 bp for short and long DNA fragments respectively). Both distributions 
have a full width at half maximum that exceeds 20 bp. This width might arise from several 
features : asymmetric unwrapping of one of the two DNA arms, AFM uncertainty and 
dispersion in octamer position. However, it is not possible with these measurements to 
determine what is the contribution of each phenomenon. Next, we can see that the distribution 
width for longer fragments seems greater. After corrections of artifacts inherent to L+/L- 
labeling (cf Supplemental Figure 2) these two position distributions are very similar showing 
that the free linker DNA does not affect either the DNA complexed length nor the positioning 
of such nucleosomes significantly.  
We have shown in this section that AFM measurements give comparable estimations 
with other methods for both the positioning and the DNA wrapping of short 601 
mononucleosomes. Furthermore, our experimental approach showed no difference in 
complexed length probability or nucleosome positioning dynamics for long and short DNA 
templates.  
For conventional and H2A.Bbd variant mononucleosomes 
In order to investigate the influence of the octamer composition on the wrapping of 
DNA around the histone octamer, a H2A.Bbd histone variant was used instead of 
conventional H2A, in order to reconstitute mono-nucleosomes on a 255 bp DNA fragment. 
The H2A.Bbd variant nucleosomes were imaged by AFM (25) and using the same analysis as 
described above, only the well positioned nucleosomes (∆L < 12 bp) were selected. Their 
DNA complexed length distribution is plotted on Fig. 2a where it is compared to conventional 
mononucleosomes reconstituted on the same 601 positioning sequence, 255 bp long, with the 
same position range selection (∆L < 12 bp).  
The average length of wrapped DNA is clearly different for the variant H2A.Bbd 
nucleosomes as the distribution peak value is L*H2A.Bbd = 130 ± 3bp instead of 
L*H2A = 146 ± 2bp for the conventional nucleosomes. Moreover the standard deviation of the 
distribution is clearly larger for the H2A.Bbd variant (σ = 41 bp to be compared to σ = 23 bp 
for the conventional nucleosomes). These differences show that the H2A.Bbd variant 
nucleosome is a more labile complex with less DNA wrapped around the octamer, in 
agreement with previous observations by AFM and cryo-EM (25). The difference in DNA 
complexed length suggests that ∼10 bp at each end of nucleosomal DNA are released from the 
octamer. Therefore, AFM allows visualizing subtle differences in the nucleosome structure. 
Finally, the DNA complexed length distribution is asymmetric for canonical 
nucleosomes. This asymmetry can be quantified by measuring their skewness 3µɶ , defined as: 
3 c c
2 22 c c
(L L )
((L L ) )
.We find 3µɶ = -0.57 ± 0.09, the negative sign meaning that 
nucleosome conformations with sub-complexed DNA, as compared to the mean value 146 bp, 
are energetically more favorable than with over-complexed DNA. This can be interpreted 
within the simple model proposed below, based on relevant structural data information (26). 
Notice that for variant nucleosomes, the complexed length distribution is nearly symmetric 
( 3µɶ ≈ 0.01 ± 0.16), and this feature will also be discussed in the modeling section. 
Simple model of DNA complexed length distribution 
It has been shown that 14 discrete contacts between DNA and histone octamer are 
responsible for the stability of the nucleosome (26).The energetic gain at these sites is made 
through electrostatic interactions and hydrogen bonding. At the length scale of the present 
analysis, the discreteness of binding sites is not relevant, and it will be replaced by a uniform 
effective adsorption energy εa< per unit length, in units of kT/bp. The finite number of binding 
sites, or equivalently the finite DNA length L* complexed through these sites (146 bp for 
canonical nucleosomes, as determined both by the present experiments and crystal structure), 
is due to the specific locations of favorable interactions located at the surface of the histone 
octamer, forming a superhelical trajectory on which DNA is complexed. DNA wrapping 
around the histone core involves additional bending penalty characterized by the energy per 
unit length : 
ε =  where Lp is the persistence length of DNA within classical linear 
elasticity and R the radius of the histone octamer. The stability of the nucleosome requires 
that the net energy per unit length is negative (energetic gain), and therefore : εb < εa< .   
The experimental distributions of DNA complexed length show that more DNA can be 
wrapped around the octamer. For these additional base pairs, the net energy per unit length 
has to be positive, due mainly to bending cost. However, to allow for the possibility of some 
residual non specific (mainly electrostatic) attractive interactions beyond the 14 binding sites, 
the energetic gain of DNA contacting the octamer surface outside of the 14 sites superhelical 
path has a different value denoted εa> . The difference εa< - εa> is then representative of the 
specificity of the 14 sites region.  
 Assuming that the energy reference is given by un-complexed straight DNA and 
octamer, the total energy for nucleosome is given by 
* * *
(sub-complexed nucleosome)
(over-complexed nucleosome)
( ) L                                   if  L <LE(L )
( ) L  + ( ) (L -L )   if  L >L
− ⋅ − ⋅
a ab b
ε ε ε ε
  (1) 
The distribution of DNA complexed length is given by cc
-E(L ) / (kT)
(L )  ∝P e . It is 
maximum for the characteristic length L*, which characterizes the region of specific contacts. 
This length may vary for canonical and variant nucleosomes. The assumptions of energy 
linearity in wrapped DNA length and of the existence of L*, lead to a double exponential 
distribution. By construction, one has the following constraints between effective energies 
εa> < εb < εa< . 
It should be kept in mind that the effective values εa>, εa< and εb are representative of 
nucleosomes adsorbed on a charged flat surface. These values might differ for nucleosomes in 
bulk solution, as discussed below. 
Extraction of the DNA complexed length parameters 
It is possible to extract some parameters from each distribution by using the physical 
model presented below, in order to interpret the experimental distribution of DNA complexed 
length. We found it more reliable to use global procedure for parameter determination, instead 
of fitting the multivariate distribution. Since we expect the DNA complexed length 
distribution to be described by a simple double-exponential model, the probability density 
function can be written as a skew-Laplace distribution which moments are calculated as : 
*2 (1 )
2 2 2
2 c c
*2 (1 ) 3 2 3
3 3/ 2 3/ 2 2 3/ 2
L 2 2
,  for L>L1
( )  and then (L L ) 4 (1 )
,  for L<L (L L ) 4 50 2 12 48 2
4 (1 )
 = = − 
 = = = − = + 
− − + −  = =
P L L
µ σ ε
ε ε ε
where L* is the most probable complexed length, ε is the relative asymmetry of the skew-
Laplace distribution and σ is the mean decay length. The distribution normalization is taken 
on full real axis as a first approximation, thus neglecting finite size effects. Given the 
experimentally determined µ
 and µ
 parameters, we extract straightforwardly the 
parameter L*, ε and σ by numerically solving the equation system (2). 
 Hence, we are able to measure without any fitting the parameters L*, ε and σ by 
calculating the first three moments µ1, µ2 and µ3 of the DNA complexed length statistical 
series. In our case we thus have : 
2(1 ) 2
 and then 
1 1 (1 )
2(1 )
 − = − = − + = − =
− − = =
specific
ads a a
To see the adequacy of this model with the experimental distribution, the function P(Lc=L) is 
drawn for the parameters extracted from the experimental data using the same 20 bp-sliding 
box protocol as for the experimental complexed length distribution (Fig.2) 
The results are summarized in table 1. The values of energies are expressed in units of 
kT per binding site, assuming 14 such sites along the 147 base pairs of DNA for canonical 
nucleosomes. Several comments are to be made on these values. First, the measured 
characteristic decay lengths corresponding to sub- (L<) and over-complexed (L>) DNA 
lengths (Table 1, (b) and (d)) are clearly higher than the intrinsic resolution of our AFM 
measurements (related to the tip size that correspond to ~ 10 bp, as checked by image analysis 
of the same naked DNA on the same surface and within the same experimental conditions -
data not shown) for both conventional and variant nucleosomes, showing therefore the 
significance of the parameters extracted here. Hence, we are able to quantify the energetic of 
both sub- and over-complexed DNA length in a mono-nucleosome. For over-complexed DNA 
length, the energy has been converted artificially into units of kT per binding site for the sake 
of comparison, although the model assumes that there are no such binding sites beyond the 14 
sites found in the crystal structure (26). If one assumes that over-complexed DNA length 
results solely from bending around the histone core (εa> = 0), the value found for εb leads to a 
persistence length Lp ~ 3.5 bp, a value definitely too small for double stranded DNA. Even 
more so, this energy is similar in amplitude to the energy of sub-complexed DNA length but 
with an opposite sign (Table 1, (c) and (e)). We conclude that it cannot simply be associated 
to a bending penalty, therefore justifying a posteriori the assumption of residual attractive 
interaction between DNA extra length and histone octamer.  
The combination of experimental asymmetry of DNA complexed length distribution 
and the simple model allows quantifying the specificity of the 14 binding sites in the 
nucleosomes (Table 1, (f)). In particular this can be interpreted as a rough estimation of non-
electrostatic contribution to adhesion energy between DNA and histone octamer.  
Comparison of model parameters extracted from data. 
These values have to be compared to other estimates reported in the literature. The net 
energetic gain per site can be compared to values extracted from experiments done in the 
group of J. Widom (68-70). The spirit of these experiments was to probe the transient 
exposure of DNA complexed length in a nucleosome by using different restriction enzymes 
acting at various well-defined sites along the DNA. The experimental results clearly 
demonstrate that DNA accessibility is strongly reduced when restriction sites are located far 
away from entry or exit of nucleosomal DNA, towards the dyad axis. From the experimental 
data, the authors extract a Boltzmann weight for different site exposures. This distribution 
should a priori be similar to the DNA complexed length distribution obtained in our work, 
except that only sub-complexed nucleosomes are probed. However, due to the use of different 
restriction enzymes with different sizes and mechanisms of action, there is an inherent 
uncertainty in the assignment of precise DNA complexed length with a free energy of the 
Boltzmann weight. In other words, only a range of energy per binding site can be extracted 
from these data. This has to be contrasted with most of previous works using Polach and 
Widom's data, which quote a single value of 2 kT per binding site (31). The range of net 
energetic gain we are able to estimate out of these data is between 0,5 to 3 kT per binding site. 
The value we extracted from our own measurements coincides therefore with the lower bound 
of this range. This might be due to the difference in the type of experiments used. 
First, our observations are made on nucleosomes adsorbed on a charged substrate. This 
might change the energetics of nucleosome opening as compared to its value in solution. A 
theoretical estimation of this change is currently under progress (Castelnovo et al, work in 
preparation). Another significant difference between Polach and Widom's experiments and 
our work is the composition of the buffer, which is known to affect the nucleosome stability. 
In particular, the buffer used for restriction enzyme assays contains more magnesium ions 
(about 10 mM MgCl2).  
The specificity of DNA binding sites on histone octamer, as determined in Table 1 (e) 
can also be compared to values extracted from X-ray experiments performed in the group of 
T.J. Richmond (71). Indeed, by counting the hydrogen bonds per binding site found in this 
structure, one can estimate the specific contribution to the binding energy. These 
contributions range between 0.8 and 2 kT per binding site (72). Our estimate for conventional 
nucleosome falls in this range (1.1 kT per binding site). 
Finally, the comparison between canonical and variant nucleosome shows that both 
the average complexed length and the energy per binding site are different. The most probable 
length L* = 127 bp (Table 1 (0)) for the variant claims for either the absence or the strong 
weakening of at least 2 binding sites. Furthermore, the energy and therefore the stability of the 
nucleosome for the remaining binding sites is reduced (εH2A.Bbd ~ 2/3 εH2A), in accordance 
with other experimental observations (16, 25, 73). We have shown in this section that a 
simple model using a linear energy for the DNA-histone interaction can be used to extract 
from the AFM data two important energetic parameters : the net energetic gain per site and 
the specific interaction between the DNA and the histone octamer per site. These values are in 
good agreement with previous biochemical and X-Ray studies done on conventional 
nucleosomes and for the first time are measured on a variant nucleosome.  
Visualization of nucleosome sliding and remodeling by 
SWI/SNF for conventional and variant nucleosomes. 
After studying the nucleosomes in their equilibrium state, the same mononucleosomes 
were visualized in the presence of the SWI/SNF remodeling factor to validate the possibility 
for this direct imaging approach to acquire new information on the mechanism and dynamics 
of nucleosome sliding. 
Centrally positioned conventional and variant mononucleosomes (Ltot = 255 bp) were 
incubated with SWI/SNF at 29°C in the presence or absence of ATP and then adsorbed on 
APTES-mica surfaces for AFM visualization. On figure 3, we report AFM images of 
mononucleosomes incubated without (Fig. 3a) and with (Fig. 3b) ATP for one hour. The 
sample containing no ATP is the control experiment to account for possible nucleosome 
thermally driven diffusion when incubated 1 h at 29 °C. The representative chosen set of 
AFM images of Fig.3 clearly shows that most of the nucleosomes are centered on the DNA 
template in the negative control (-ATP) whereas they rather exhibit end-position when 
SWI/SNF and ATP are present.  
On AFM images, SWI/SNF motor is sometimes visible as a very large proteic 
complex, and if still attached to nucleosome prevents any image analysis of such objects. Our 
protocol does not include removing of SWI/SNF before deposition, even if by diluting the 
nucleosome/motor mix, one could expect detaching of some motors. Therefore, the motor per 
nucleosome ratio used in the sliding experiments is kept low with respect to biochemical 
assays (55) (roughly five time less SWI/SNF per nucleosome). 
Using the same type of image analysis we were able to reconstruct 2D histograms 
Lc/∆L (using a 2D sliding box as described in the Material and Methods section) at various 
time steps during nucleosome sliding : 0 (-ATP), 20 min and 1 hour (Fig. 4). We first notice 
that in the absence of ATP, SWI/SNF has apparently no effect on the Lc/∆L map. The 2D 
distribution exhibits a single peak corresponding to the canonical nucleosome positioned as 
expected from the DNA template (α state). As a function of time in the presence of 
remodeling complex and ATP, new states appear : (β) corresponds to an over-complexed 
nucleosome having the same mean position ∆L value as (α); this state could result from the 
capture of extra DNA (a loop of ~ 40 bp) inside the NCP induced by SWI/SNF. (β)-state is 
spread in the ∆L direction showing that this extra complexed DNA length (~ 40 bp) seems to 
exist for various positions of the nucleosome (0 < ∆L < 30 bp). (γ) is the slided end-positioned 
nucleosome (∆L ~ 50 bp) having slightly less DNA wrapped around the histone octamer 
(Lc ~ 125 bp). The ∆L distance separating (α) and (γ) states is close to the Lc distance 
between (β) and (α) states, meaning that the slided (γ)-state most likely results from the 
release of the (β)-state DNA loop (~ 40 bp). The fact that slided nucleosomes are sub-
complexed i.e. their dyad has been moved beyond the expected end-position, has already been 
observed in other biochemical studies (74). Similarly, the anisotropic spreading of the 
(γ)-peak towards higher ∆L and lower Lc is also consistent with this feature. We cannot 
exclude that a finite size effect of the DNA template could account for this feature. Finally, 
(δ) is a wide state with a sub-complexed Lc ~ 75 bp, that could correspond to a tetrasome or 
hexasome. This state could be due to the loss of one wrapped DNA turn either from the α 
state or the (γ)-state. Nevertheless, one could notice that the (δ)-state is missing on the ‘+ATP 
20 min’ map (Fig. 4b) where only few nucleosomes have been slided (weak γ peak) whereas 
(δ)-state nucleosomes are clearly visible on Fig. 4c (‘+ATP 1 h’). This tends to show that (δ)-
state nucleosomes more likely arise from the loss of one DNA turn of the end-positioned 
nucleosomes ((γ)-state).  
We have seen that the 2D-mapping of nucleosome position and DNA complexed 
length allows characterizing the new states resulting from the ATP-dependent action of 
SWI/SNF on our 601 nucleosomes : an over-complexed state close to the 601 template center 
(β), a slided state (γ) and a sub-complexed state (δ). 
Again, more information can be gained by appropriate projections of these 2D-
histograms. Nucleosomes having their DNA complexed length in the range L* ± σLc were 
selected (L* and σLc are respectively the maximum value and the standard deviation of the 
corresponding complexed length distribution) and their position distribution is plotted on 
Fig. 5a. For conventional nucleosomes with SWI/SNF but no ATP, the distributions obtained 
for nucleosome position (Fig. 5a) and DNA complexed length (Fig. 5b) are very similar to the 
case without any remodeling complex (Fig. 1b), showing no effect of thermally driven 
diffusion of mononucleosomes reconstituted on 601 positioning sequence in our conditions.  
When incubation is increased in the presence of ATP (20 minutes and 1 hour), the 
position distribution of conventional nucleosomes is clearly changed (Fig. 5a). Indeed, as a 
function of incubation time, a second peak appears corresponding to the end-positioned 
nucleosomes (∆L ~ 50 bp, cf (γ)−state in Fig. 4c). After one hour of SWI/SNF action in 
presence of ATP the second peak height has increased at the expense of the primary peak. 
This corresponds to the situation were one third of the mono-nucleosomes are positioned at 
the end of the DNA template. It is interesting to note that during the remodeling factor action 
we do not see any significant increase in the amount of nucleosomes in an intermediate 
position (20 bp < ∆L < 40 bp). This provides experimental evidence that this remodeling 
factor moves centrally positioned nucleosomes directly to the end of our short DNA template.  
Mainly, two situations can explain the bimodal position distribution of nucleosomes 
after the action of SWI/SNF. The first hypothesis is that the SWI/SNF complex is a 
processive molecular motor. As it will not detach from the nucleosome before it reaches the 
end of the DNA template, the elementary step of the SWI/SNF induced sliding might not be 
accessible. Indeed, in our experimental conditions, only nucleosomes without SWI/SNF 
complex attached can be analyzed. The other possibility is that SWI/SNF is weakly 
processive (SWI/SNF turnover rate is unknown) but with an elementary step of the order of 
50 bp, which corresponds to the value measured by us and other approaches (75-77), and 
happens to be the length of free DNA arms in our case. Therefore, a single step would be 
enough for the motor to slide a nucleosome to an end-position and release the complex.  
Nevertheless, another mechanism cannot be excluded by our data, where SWI/SNF 
action would consist of octamer destabilization followed by thermally driven diffusion 
towards the end-positioned entropically favored. In this situation, ATP-hydrolysis would only 
be involved in the nucleosome ‘destabilization’ step.  
In Fig. 5b, we show projections of the previous 2D-histograms along the DNA 
complexed length axis without any selection on their position. For conventional nucleosomes 
in the presence of SWI/SNF but no ATP, the complexed length distribution is similar to case 
with neither SWI/SNF nor ATP. However, the former distribution is larger due to the 
contribution of different nucleosome positioning. Then after 20 min, the distribution is 
broader (roughly twice) and shifted towards higher Lc. This might be attributed to the 
contributions of the different states (β, γ, δ) identified in the Fig. 4b/c. The increase in Lc 
mean value is likely due to the statistical weight of the over-complexed (β)-state. 
The same sliding experiment was performed on H2A.Bbd variant nucleosomes in 
absence and in presence of ATP and analyzed through the projection of the 2D-histogram 
Lc/∆L. No significant effect of SWI/SNF complexes in presence of ATP on the position 
distribution of H2A.Bbd variant nucleosomes is observed (Fig. 5c). This corroborates 
previous findings using biochemical sliding assay done on 5S and 601 positioning sequence 
(55). However in AFM measurements, the full position distribution is accessed directly with a 
resolution better than 10 bp (the size of AFM tip). This variant nucleosome sliding assay 
shows the reproducibility of our experimental approach as not only the position distribution 
mean value is constant during one hour in the presence of SWI/SNF and ATP, but also the 
complete position distribution remains constant (Fig. 5c). Similarly, SWI/SNF in presence of 
ATP does not seem to influence the DNA complexed length distribution of H2A.Bbd 
nucleosomes (Fig. 5d). 
CONCLUSION 
In summary, we have shown that AFM combined with a systematic computer analysis 
is a powerful tool to determine the structure of conventional and variant mononucleosomes at 
equilibrium and after the action of ATP-dependent cellular machineries. With this technique 
we have quantified simultaneously two important and closely coupled variables : the DNA 
complexed length and the position of mono-nucleosomes along the 601 DNA template. For 
each of these two distributions, the most probable value is in perfect agreement with 
measurements done by other methods that give access to one of these two parameters only. In 
addition, to explain the experimental complexed length distribution, we have developed a 
simple model that uses the experimental shape of DNA complexed length distributions to 
quantify the interaction of DNA with histones. With this model, we extract both the net 
energetic gain for sub-complexed nucleosomes and the estimation of the non-electrostatic 
contribution to the adhesion energy between DNA and histone octamer . 
We further show that H2A.Bbd variant and conventional nucleosomes exhibit clear 
differences in DNA complexed length and in their ability to be slided by SWI/SNF. Indeed, 
these variant nucleosomes organize less DNA on average than conventional nucleosomes, and 
present larger opening and closing fluctuations. Moreover, the whole position distribution as 
well as complexed length distribution remain unchanged showing H2A.Bbd variant is neither 
displaced nor remodeled by SWI/SNF complex. 
Finally, we have plotted Lc/∆L as a 2D map of the nucleosome states. This 
representation is well suited to highlight the various nucleosome states that appear during the 
SWI/SNF action. For example, as a function of time, we have evidenced the formation of an 
over-complexed state followed by the appearance of a slided state. More quantitative 
information can be obtained by appropriate projections of the 2D-histograms, as for instance 
the bimodal position distribution induced by SWI/SNF sliding on conventional nucleosomes, 
suggesting two possible scenarii : a processive action of the molecular motor (no intermediate 
position visualized) or an elementary stepping length (~ 40 bp) of the size of the free DNA 
arms (~ 50 bp). The short length of DNA templates and lack of directionality in our position 
analysis prevent us from discriminating between these two hypotheses, and further 
experiments on long oriented mononucleosomes are needed to get more insights into the 
molecular mechanism of SWI/SNF action. The present results as well as preliminary data on 
longer oriented templates prove nevertheless that this extension will provide useful 
information on remodeling mechanisms of SWI/SNF.  
 A further perspective of this AFM study will be to test the effect of the flanking DNA 
sequences on the conformation and dynamics of 601 nucleosomes. Nevertheless, in order to 
test sequence effect, nucleosomes should be reconstituted on less positioning sequences (5S 
rDNA for example) or non-positioning sequences, but this will complicate significantly the 
nucleosome sliding analysis as the initial position distribution of the nucleosome is expected 
to be broader in this case. 
ACKNOWLEDGMENTS 
We thank Dimitar Angelov and Hervé Ménoni for various forms of help with 
nucleosome reconstitution and sliding assays and Cécile-Marie Doyen for producing 
H2A.Bbd variant histones. We are grateful to Stefan Dimitrov, Dimitar Angelov, Françoise 
Argoul, Alain Arneodo, Cédric Vaillant and Phillipe Bouvet for fruitful discussions. We thank 
Ali Hamiche for providing us with the ySWI/SNF complex. P.St-J. acknowledges CRSNG for 
financial support. This work was supported by the CPER ‘Nouvelles Approches Physiques 
des Sciences du Vivant’. 
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TABLE 1 
<Lc>  
(bp) 
decay length L< 
(bp) 
εεεεb - εεεεa< 
(kT per site) 
decay length L> 
(bp)  
εεεεb - εεεεa> 
(kT per site)  
εεεεa< - εεεεa> 
(kT per site)  
conventional 
nucleosome 146 ± 2 22 ± 1.6 -0.479 ± 0.045 17 ± 1.4 0.61 ± 0.064 1.1 ± 0.072 
variant 
nucleosome 
127 ± 3 31 ± 1.5 -0.33 ± 0.022 27 ± 1.5 0.39 ± 0.026 0.72 ± 0.021 
site exposure 
model (g) 
  -3 <...< -0.5    
crystal 
structure (h) 
147     0.8<…<2 
Caption Table1 : Summary of model parameters extracted from experimental data as 
explained in Materials and methods. All energies are expressed in units of kT per binding site. 
DNA lengths are expressed in bp. (a) Average complexed length (b) Characteristic length L< 
of exponential decay towards sub-complexed DNA length. (c) Energy per binding site (1/ L<) 
for sub-complexed DNA length. (d) Characteristic length L> of exponential decay, towards 
over-complexed DNA length. (e) Energy per binding site (1/L>) for over-complexed DNA 
length. (f)Asymmetry of adhesion energy per binding site between sub- and over-complexed 
DNA length. (g) Range of values extracted from Polach and Widom (27, 69) data using the 
site exposure model. (h) Range of values extracted from Davey and Richmond (71) data using 
X-ray crystal structure of the nuclear core particle. Uncertainty values are determined using 
the central limit theorem and a propagation of uncertainty calculus detailed in supplemental 
data. N(H2A conventional) = 301 nucleosomes. N(H2A.Bbd variant) = 252 nucleosomes. 
FIGURE CAPTIONS 
Figure 1 : AFM visualization of centered mononucleosomes with short and long arms. 
(a) AFM topography image of mono-nucleosomes reconstituted on 356 bp 601 positioning 
sequence. Color scale : from 0 to 1.5 nm. X/Y scale bar : 100 nm. (b) Zoom in the AFM 
topography image of a centered mono-nucleosome and the result of the image analysis. Black 
line : contour of the mono-nucleosome. Blue point : centroid of the histone octamer. Blue dot 
circle : excluded area of the histone octamer. Blue line : skeletons of the free DNA arms. 
Color scale : from 0 to 1.5 nm. X/Y Scale bar : 20 nm. The longest arm is named L+ and the 
shortest L-. DNA complexed length is deduced by Lc = Ltot - L- - L+ where Ltot is in this case 
356 bp. The position of the nucleosome relatively to the center of the sequence is calculated 
by ∆L = (L+ - L-)/2. (c) 2D histogram Lc/∆L representing the DNA complexed length Lc along 
with the nucleosome position ∆L for a short DNA fragment of 255 bp 
(N = 702 nucleosomes). (d) Probability density function of the DNA complexed length Lc for 
a short DNA fragment (255 bp, purple line) and for a long DNA fragment (356 bp, blue line) 
obtained by selecting the well positioned nucleosomes (0 < ∆L < 12 bp and 12 < ∆L < 32 bp 
for the short and long fragments respectively) and projecting the 2D map along the y-axis. (e) 
Probability density function of the ∆L nucleosome position for a short DNA fragment 
(255 bp, purple line) and for a long DNA fragment (356 bp, blue line) obtained by selecting 
nucleosomes having their DNA complexed length Lc in the range 123 bp < Lc < 169 bp for 
both fragments, and projecting the 2D map along the x-axis.  
Figure 2 : AFM Visualization of centered H2A.Bbd variant and H2A conventional 
mononucleosome. 
(a) Probability density function of the DNA complexed length Lc for a short DNA fragment 
(255 bp) with conventional H2A (solid thick line) and with variant H2A.Bbd (dotted thick 
line) nucleosome. Simple model for conventional and variant nucleosomes (respectively solid 
and dashed thin lines). (b) Description of the model used to measure the DNA-histone 
adsorption energies per bp (εa< and εa>) and the DNA bending energy per bp (εb) (dotted line). 
Representation of the model using the 20bp-sliding-box procedure (dotted dashed line). L* 
corresponds to the most probable DNA complexed length of the distribution. 
Figure 3 : AFM Visualization of the sliding of centered mononucleosomes by the 
remodeling complex SWI/SNF.  
AFM topography image of mononucleosomes reconstituted on 255 bp 601 positioning 
sequence, incubated at 29°C with SWI/SNF for one hour (a) in the absence and (b) in the 
presence of ATP. Color scale : from 0 to 1.5 nm. X/Y Scale bar : 150 nm. Zoom in the AFM 
topography image of a (c) centered mononucleosome and (d) end-positioned 
mononucleosome the result of the image analysis. Black line : contour of the mono-
nucleosome. Blue point : centroid of the histone octamer. Blue line : skeletons of the free 
DNA arms. Color scale : from 0 to 1.5 nm. X/Y Scale bar : 20 nm.  
Figure 4 : Evolution of nucleosome Lc/∆∆∆∆L map during nucleosome sliding by SWI/SNF 
complex for conventional nucleosome.    
2D histogram Lc/∆L representing the DNA complexed length Lc along with the nucleosome 
position ∆L for a conventional nucleosome reconstituted on a short DNA fragment (255 bp) 
in the presence of remodeling complex SWI/SNF (a) without ATP (1h at 29°C) (b) with ATP 
(20 min at 29°C) and (c) with ATP (1h at 29°C). (d) Representation of the nucleosome Lc/∆L 
states for (α), (β), (χ) and (δ) positions as pointed on the 2D maps. N(-ATP, 1h at 
29°C) = 692 nucleosomes, N(+ATP, 20 min at 29°C) = 245 nucleosomes, N(+ATP, 1h at 
29°C) = 655 nucleosomes. 
Figure 5 : Evolution of nucleosome position and DNA complexed length distributions 
during nucleosome sliding by SWI/SNF complex, for conventional and variant 
nucleosome. 
Nucleosome position ∆L (a) and DNA complexed length Lc (b) distributions as a function of 
time (0, 20 min, 1 hour) in the presence of SWI/SNF, for conventional mono-nucleosomes 
reconstituted on 255 bp long 601 positioning sequence. Nucleosome position ∆L (c) and DNA 
complexed length Lc (d) distributions as a function of time (0, 1 hour) in the presence of 
SWI/SNF, for H2A.Bbd variant mono-nucleosomes reconstituted on the same DNA template. 
The zero time is given by the control in the absence of ATP (solid purple line). For each ∆L 
position distribution, only nucleosomes having their complexed length in the range Lc
* ± σLc 
are selected. For the sake of figure clarity, error bars are only depicted on 2 distributions of 
graph (a). 
Lc = Ltot - L-- L+  
∆∆∆∆L = (L+-L-) / 2
Figure 1
(a)(a) (b)(b)
L-=86bp
L+=130bp
Ltot = 255 bp,
0 < ∆L< 12 bp
Ltot = 356 bp,
12 < ∆L< 32 bp
0 0.01 0.02
Probability
Ltot = 255 bp,
123 < Lc  <169 bp
123 < Lc  <169 bp
Ltot = 356 bp,
Lc (bp)
∆∆ ∆∆
50 150 250200100
12e-4
probability scale
Figure 2
Lcomplexed
c( ) L( )   b aP ε εα <+ ⋅ *c cL e if  L < L c( ) L( )   b aP ε εα >+ ⋅ *c cL e if  L > L
-50 0 50 100 150 200 250 300
0.005
0.015
complexed
H2A, L
=255bp, ∆L<12bp
H2A-Bbd, L
=255bp, ∆L<12bp
Model H2A
Model H2A.Bbd
+ ATP + SWI/SNF
- ATP + SWI/SNF
Figure 3
Figure 4
((((a))))
((((b))))
(α)(α)
(α)(α)
(α)(α) (β)(β)
(γ)(γ)
(δ)(δ)
LLLLcccc (bp)(bp)(bp)(bp)
(γ)(γ)
147bp (601)
12e-4
probability 
scale
7.5e-4
2.5e-4
7.5e-4
2.5e-4
0 50 100 150 200 250 300
0.002
0.004
0.006
0.008
0.012
-ATP t=1h
+ATP t=20min
+ATP t=1h
H2A nucleosomes
0  25 50 75 100
0.005
0.015
∆∆∆∆ L (bp)
-ATP 1h
+ATP 1h
H2A.Bbd nucleosomes (d)
0 50 100 150 200 250 300
0.002
0.004
0.006
0.008
0.012
-ATP t=1h
+ATP t=1h
H2A.Bbd nucleosomes
(a) H2A nucleosomes
0  25 50 75 100
0.005
0.015
∆∆∆∆L (bp)
-ATP,  t=1h
+ATP,  t=20min
+ATP,  t=1h
Figure 5
