Dissecting DNA-Histone Interactions in the Nucleosome by Molecular
Dynamics Simulations of DNA Unwrapping
Ramona Ettig,†Nick Kepper,†Rene Stehr,‡Gero Wedemann,‡and Karsten Rippe†*
†Deutsches Krebsforschungszentrum (DKFZ) and BioQuant, Research Group Genome Organization & Function, Heidelberg, Germany; and
‡University of Applied Sciences Stralsund, Competence Center Bioinformatics, Stralsund, Germany
otes and regulates the access of protein factors to the DNA. We performed molecular dynamics simulations of the nucleosome
in explicit water to study the dynamics of its histone-DNA interactions. A high-resolution histone-DNA interaction map was
derived that revealed a five-nucleotide periodicity, in which the two DNA strands of the double helix made alternating contacts.
On the 100-ns timescale, the histone tails mostly maintained their initial positions relative to the DNA, and the spontaneous un-
wrapping of DNA was limited to 1–2 basepairs. In steered molecular dynamics simulations, external forces were applied to the
linker DNA to investigate the unwrapping pathway of the nucleosomal DNA. In comparison with a nucleosome without the
unstructured N-terminal histone tails, the following findings were obtained: 1), Two main barriers during unwrapping were iden-
tified at DNA position 570 and 545 basepairs relative to the central DNA basepair at the dyad axis. 2), DNA interactions of the
histone H3 N-terminus and the histone H2A C-terminus opposed the initiation of unwrapping. 3), The N-terminal tails of H2A,
H2B, and H4 counteracted the unwrapping process at later stages and were essential determinants of nucleosome dynamics.
Our detailed analysis of DNA-histone interactions revealed molecular mechanisms for modulating access to nucleosomal DNA
via conformational rearrangements of its structure.
The nucleosome complex of DNA wrapped around a histone protein octamer organizes the genome of eukary-
The nucleosome is the fundamental unit of chromatin in
H2A, H2B, H3, and H4, and 146/147 basepairs (bp) of DNA
(1,2). The DNA stably contacts the surface of the histone
protein octamer core in a left-handed superhelix of almost
two turns. Histone proteins consist of a globular part formed
by threewell-structured a-helices and the histone tails. These
variable conformations. Removal of the histone tails leads to
some increase of nucleosome flexibility and affects the
binding of other proteins to the nucleosome and/or its associ-
ated DNA (3–5). The interactions of the unstructured histone
N-terminal tails are modulated by posttranslational modifica-
tions like acetylation, methylation, and phosphorylation at
in a dynamic manner by specific enzymes (6,7). Histone tails
interact with the posttranslationally modified histone state. In
addition, acetylation of histone lysines has a direct effect on
the stability of the nucleosome core particle, and on its
higher-order interactions because the positively charged
lysine is neutralized in the acetylated state (8).
The dynamics of the nucleosome in terms of spontaneous
DNA unwrapping or breathing were studied by various
retically how proteins like restriction enzymes or RNA poly-
merases can access nucleosomal DNA and depend on the
spontaneous unwrapping of DNA (9–11). The transition
Theseyielded lifetimes ofseveralsecondsfor the closedstate
that were interrupted by open periods of a few tenths of
a second, in which up to 80 bp of nucleosomal DNA were
exposed. Finally, the unwrapping of nucleosomal DNA is
referred to as remodeling complexes. They mediate the ATP-
dependent translocations of nucleosomes along the DNAvia
(16,17).Thus,understandingthe energeticsofthe DNAinter-
action with the histone octamer is essential for dissecting
cellular processes that control unwrapping of nucleosomal
tion between the DNA and histone protein can be directly
investigated in force spectroscopy experiments (18–23).
To relate the experimentally observed nucleosome dy-
dynamics (MD) studies are ideally suited (24–27). They
allow for a detailed comparison of free DNA with DNA
bound to the histone octamer, and an analysis in terms of
dynamic DNA double helix features and superhelix configu-
ration (24,26,28). MD studies of systems in the size of the
nucleosome already require large computational resources
for simulations of tens of nanoseconds. However, with
respect to nucleosome dynamics, many processes occur at
the microsecond to second timescale (13,15). By applying
Submitted January 20, 2011, and accepted for publication July 27, 2011.
Editor: Laura Finzi.
? 2011 by the Biophysical Society
Biophysical Journal Volume 101 October 2011 1999–20081999
an external force in so-called steered molecular dynamics
state can be induced and compared to results from single-
molecule force spectroscopy experiments (29–31). Here,
we have performed all-atom MD and SMD simulations of
a solvated nucleosome with and without the N-terminal
histone tails to investigate the process of unwrapping DNA
from the histone octamer. Our findings reveal details of the
dynamic protein-DNA interactions at atomic resolution that
govern the accessibility of nucleosomal DNA. This has
scription factors to DNA sites occupied by a nucleosome.
MATERIALS AND METHODS
MD and SMD simulations were based on the structure of nucleosome and
linker DNA in the tetranucleosome crystal structure 1ZBB (32) that had
undergone minimization and had been solvated and equilibrated for 2 ns or
50 ns (33) (see structure NUC, Fig. S1 A, in the Supporting Material). For
simulations of the tailless nucleosome structure the N-terminal tails were
removed from the 50-ns equilibrated complete nucleosome structure so that
only amino acids 41–135 (H3), 25–102 (H4), 17–128 (H2A), and 32–122
(H2B) remained (structure NUCDtail). DNA sequence effects were studied
in comparison to an additional nucleosome crystal structure with an adenine
dA16$dT16insert (based on coordinates PDB 2fj7, and termed structure
NUC_A16) In addition, a structure referred to as NUC_loop was evaluated
that had a central DNA loop of 20 bp (see Fig. S1 B). Simulations were con-
ducted with the NAMD 2.6 (34) and AMBER 10.0 (35) software packages.
Details of the MD and SMD simulations and data analysis are described in
the Methods section in the Supporting Material.
DNA-histone interactions occur with
a five-nucleotide periodicity
To investigate the interactions between DNA and histone
trations of monovalent ions. In Fig. 1 A, the corresponding
time-dependent DNA-histone interactions are shown. Addi-
tionally, the dynamics of DNA-histone contacts during
a 120-ns MD simulation of a nucleosome structure with an
internal DNA loop were investigated (see Fig. S2). For the
NUC structure, the Fourier spectrum of the time-averaged
interactions of the histone octamer and the DNA sequence
was calculated (Fig. 1 B). It revealed a peak at 0.19/bp, corre-
the interactions of the two DNA strands of the double-helix
separately, a ~10-nucleotide periodicity for each DNA strand
with a ~5.5-nucleotide phase shift between the strands was
the DNA sequence on the observed periodicity, the Fourier
spectra of a nucleosome with an insert of dA16at bp ?35
was also calculated. This yielded the same results for the
interaction periodicity, albeit with weaker histone-DNA
interactions in the region of the adenine tract (see Fig. S3).
Histone tail interactions stabilize the nucleosome
and can cause an asymmetric DNA interaction
The images in Fig. 1 A illustrate the temporal evolution of
interactions between DNA and histone proteins. From
a comparison of the complete nucleosome with a nucleo-
some without the N-terminal histone tails, the contribution
of the tails can be extracted. As expected, the interactions
between DNA and the globular parts of the histone octamer
were symmetric relative to the dyad axis. However, due to
the variability of contacts made by the flexible tails, the
interaction pattern became asymmetric. Thus, factors that
direct the histone tails to certain DNA contact regions could
induce a preference for unwrapping from one linker DNA
side. It appears likely that the tail-DNA interaction pattern
will become symmetric on longer timescales due to
sampling over the different conformations. However, on
the 100-ns timescale, no large rearrangements of the histone
tails were observed in the MD simulation (Fig. 1 A, and see
Fig. S2). The only exception was a relocation of the H2A
C-terminus that is depicted in Fig. S4. The spontaneous
opening of the DNA-histone interactions at the entry-exit
site of the DNA in the nucleosome as well as at the start/
end of the looped region comprised only 1–2 bp during
the observation period (Fig. 1 A, and see Fig. S2, Fig. S5,
Fig. S6, and Movie S1 in the Supporting Material).
Unwrapping nucleosomal DNA by applying
an external force reveals five phases of DNA
unwrapping with two main energy barriers
at ±70 bp and ±45 bp
To investigate the unwrapping of DNA from the histone
octamer, SMD simulations were conducted. One end of the
nucleosomal DNA was fixed, and a harmonic potential
moving at constant velocity was applied to the opposite
DNA end (Fig. 2). Due to the limitations of the available
studies, important information on the most relevant energy
barriers as well as the reaction pathway can be derived
despite this limitation (29,36–38). The application of the
SMD approach to dissect the DNA unwrapping process
from the nucleosome identified five characteristic phases
and two main energy barriers (Figs. 2 and 3, and see Movie
S2 and Movie S3).
Phase I: bending of linker DNA
At the entry-exit site of the nucleosome, the DNA became
bent into the direction of applied forces. Without breaking
contacts with the histone residues, the nucleosome aligned
and the DNA elongated by rearranging bases (tilting and
Biophysical Journal 101(8) 1999–2008
2000Ettig et al.
untwisting). Interactions of the globular histones and the
DNA at position 570 bp were enhanced by binding of the
C-terminal H2A and N-terminal H3 tails. These defined
a first barrier toward unwrapping.
Phase II: unwrapping of the outer DNA turn
Upon further DNA unwrapping, the barrier at 570 bp was
broken and DNA segments were released in~5 bpstepsuntil
octamer core. The outer turn comprised ~67 bp, i.e., the
region from 573 bp to 540 bp.
Phase III: protein core rotation
To enable further DNA unwrapping, the protein core rotated
around the dyad axis. The extension of the DNA ends re-
sulted mostly from the rotation itself and from reorganiza-
tion of already unwrapped DNA.
Phase IV: inner DNA turn opening
The DNA unwrapping process proceeded differently for the
complete and tailless nucleosomes. For both structures, a
second barrier for further unwrapping of DNAwas apparent
-70-60 -50-40-30 -20-100 1020 30 405060 70
-70 -60-50 -40-30 -20-10
DNA Position (bp)
0 10 2030 40506070
DNA Position (bp)
H2B H4H3 & H2A H3 & H2A H2A H4 H2B
H3 & H2A
Spatial Periodicity (1/bp)
H2BH4 H3 & H2AH3 & H2A H2A H4H2B
H3 & H2A
action maps. (A) Temporal evolution of the
histone-DNA electrostatic and van der Waals inter-
actions in a nucleosome calculated for two
20-ns MD simulation trajectories. The interaction
strength increases (from white to black). The
DNA position numbering refers to the central base
pair 0 at the nucleosomal dyad axis. The three
panels in each of the two MD simulations differ
with respect to the histone residues that were taken
into account for calculating interaction energies
with the DNA. They show the complete nucleo-
some (complete, top panel), the interactions
without the N-terminal tails (globular, middle
panel), and the isolated tail contributions (tails,
bottom panel). The colored boxes indicate the
specific histones that are involved in the DNA
interactions in this region (H3 and C-terminal
H2A, yellow; N-terminal H2A, red; H2B, blue;
and H4, green). The first MD simulation shown
(top panel) was conducted with a start configura-
tion of a nucleosome that was derived from the
crystal structure (32) but had been already equili-
brated for 50 ns (33). The nucleosome start struc-
ture of the second MD simulation shown (bottom
panel) was derived from the same crystal structure,
minimized, and equilibrated for only 2 ns. (B) The
temporal mean energy values of histone-DNA
interactions for the entire MD simulation trajectory
are plotted with respect to the DNA position for the
complete (blue line) and the tailless (red line)
nucleosome. Relative to the nucleosome dyad
axis, the red line was much more symmetric than
the blue one, demonstrating that the histone tails
have a strong influence on the interaction pattern
and the interaction symmetry within the nucleo-
some. (C) Fourier spectra for complete and tailless
nucleosome interaction maps calculated with the
data from panel B. When the unstructured histone
tails were excluded, a pronounced frequency peak
appeared at 0.19/bp that corresponds to a 5.1-bp
High resolution DNA-histone inter-
Biophysical Journal 101(8) 1999–2008
Nucleosome Unwrapping Simulation2001
DNA on one side of the nucleosome was fixed (represented by the gray sphere), whereas the C10atom of the last nucleotide of the opposite linker DNAwas
moved alongthe indicated directionwith anadditionalharmonicpotential. Snapshotsofthe complete (leftpanel) andthe tailless(right panel) nucleosomesat
the indicated times of the SMD simulations are displayed. The complete trajectory is shown in Movie S2 and Movie S3. (Color code: H2B, blue; H2A, red;
H3, yellow; H4, green; and DNA, gray.)
Intermediate structures formed during the DNA unwrapping process of a complete and a tailless nucleosome. The last nucleotide in the linker
Biophysical Journal 101(8) 1999–2008
2002Ettig et al.
around bp position 545. This was more pronounced for the
NUC structure due to the DNA binding of H2A and H2B
N-terminal tails. In addition, a partial disassembly of the
histone core protein secondary structure occurred. It
involved the disruption of interactions between the two
H2A$H2B dimers due to changes of the relative location
of a-helices in the histone fold and their partial unwinding.
The latter process was apparent in both H2A histones in the
first N-terminal a-helix (at ~62 nm/~75 nm extension, t ¼
~13.4 ns/~16.1 ns) as well as in the second (at ~71 nm/
~78 nm extension, t ¼ ~15.1 ns/~16.7 ns).
Both H2Bs displayed an opening of the angle between the
two C-terminal a-helices at extension ~92/~113 nm and t ¼
~19.5 ns/~21.5 ns. This was more pronounced for the H2B
widening of the angle between the first N-terminal a-helices
(extension ~96.5 nm, t ¼ ~21.4 ns). In addition, the
H2A$H2B dimers were shifted slightly relative to the
helix unwound (extension ~101 nm, t ¼ ~20.2 ns), whereas
the other H3 and both H4s did not change their folding. For
DNAunwrappingfrom the tailless nucleosome,noreorgani-
zation of the histone octamer structure was apparent. The
opening of the inner DNA turn comprised the disruption of
the DNA-core histone interactions at 545 bp.
Phase V: unwrapping of inner DNA turn
DNA-core histone contacts were successively disrupted
until the DNAwas completely straightened along the direc-
tion of the applied force. Residual contacts between the
globular histone domains and the stretched DNA remained
only at the former dyad axis. In addition, some histone
tail-DNA interactions persisted.
The N-terminal histone tails and the H2A
C-terminal tail counteract DNA dissociation
from the nucleosome
The H3 N-terminus and the H2A C-terminus contributed to
the first barrier toward nucleosome unwrapping because
they interacted with the DNA at the entry-exit site (see
Fig. S4 and Fig. S7). Both tails were located in the minor
groove and bind DNA via a variety of electrostatic and
van der Waals interactions as well as hydrogen bonds. After
linker DNA bending to ~90?relative to the nucleosomal
DNA, the structure extended due to DNA stretching with
partial DNA unstacking. Because the latter process is ener-
getically unfavorable, the H3 and H2A tails must provide
a significant contribution to the histone DNA interaction
that prevented DNA unwrapping during a ~4-ns time period
at the 570 bp boundary (Fig. 3 A). At 540 bp, the
N-terminal H2A and H2B tails stabilized the inner DNA
In response to the applied potential, the N-terminal
a-helices of H2A started to break open after ~16 ns. This
transition allowed it to maintain DNA contacts with the
H2A and H2B tails (Fig. 2, Fig. 3 A, and Movie S2). Inter-
estingly, the histone tails remained associated with the DNA
during the entire 24-ns simulation period up to the point
when the DNAwas fully unwound from the histone octamer
core. Their flexible conformation accommodated the signif-
icant DNA extension in the SMD experiments. The above
contributions of the histone tails can be identified from the
behavior of the NUCDtail structure in the SMD simulations
(Fig. 2, Fig. 3 B, and Movie S3), which displayed the
following differences as compared to the complete nucleo-
some: 1), The first breakage of histone-DNA contacts
at ?70 bp occurred before 3.8 ns (Fig. 2, indicated by the
arrow). 2), After breaking the contacts at 570, the DNA
dissociated more continuously from the protein core. 3),
The second energy barrier toward unwrapping at 540 bp
was much less pronounced. 4), DNA-histone interactions re-
mained only over a small region of ~20 bp in the unwrapped
structure (Fig. 3 B).
Differences in the stability of the inner and outer
DNA turn are partly due to additional histone
tail-DNA interactions when the outer turn
In experimental studies, an apparent higher stability of the
inner DNA turn has been reported (19,22,23,39). As
proposed previously, this could include a contribution of
the histone tails (39,40): While the tails are bound to both
Tailless nucleosomeComplete nucleosome
-60 -40 -20
DNA Position (bp)
DNA Position (bp)
-60 -40 -20
actions between DNA and histones during the unwrapping simulations.
Changes of relative interaction strength from strong (black) to no-interac-
tion (white) during the unwrapping simulations are shown. Contacts broke
first between the histone protein residues and DNA bp closest to the DNA
entry-exit site of the nucleosome. Regions with differences in disruption of
interactions between complete and tailless nucleosomes were found around
bp ?18 to ?25, ?35 to ?42, and 20 to 35 where the histone tails main-
tained contacts even with the completely unwrapped DNA. (A) Complete
nucleosome. (B) Tailless NUCDtail structure.
Temporal evolution of electrostatic and van der Waals inter-
Biophysical Journal 101(8) 1999–2008
Nucleosome Unwrapping Simulation2003
DNA turns in a complete nucleosome, they might reposition
to the inner turn as soon as the outer turn is unwrapped. We
calculated the DNA interactions of all histone tails sepa-
rately over the unwrapping trajectory. An ~20% fraction
of histone tail interactions rearranged from the outer to the
inner DNA turn during unwrapping of the outer DNA turn
at ~12 ns, resulting in a moderate stabilization of the inner
DNA turn (Fig. S8). The most relevant histone tail rear-
rangements involved in this process took place for the
N-terminal tails of H2A, H2B, and H4. For example, after
~15 ns, the DNA interaction of the N-terminal tail of one
histone H2A (top-left image in Fig. S8) increased as indi-
cated by a change of the color coding to dark blue and relo-
cated from a position at ~þ42 bp toward þ30 bp. In
addition, both H2B N-termini displayed significant move-
ments in the >12-ns time regime, and to a somewhat lesser
extent the same was observed for the H4 tails.
In addition to changes of the histone tail location, the two
DNA turns might repel each other while being wrapped
around the histone proteins, and the inner turn might be
stabilized by a reduced repulsion when the outer DNA
turn is unwrapped (39,40). Our analysis of the DNA-DNA
strand repulsion revealed a nonhomogeneous relatively
low repulsion of 1–10% of the total calculated interaction
energy at only a few bp positions (see Fig. S9). If averaged
over the entire nucleosome, this value was reduced to
a minor fraction of the total DNA-histone interaction
energy. Accordingly, the effect of DNA repulsion on desta-
bilizing the outer DNA turn appears to be negligible.
The force extension curve of unwrapping
nucleosomal DNA results from a complex overlay
of stretching and disruption events
Fig. 4 shows force-extension curves computed from the un-
wrapping simulations of the complete and tailless nucleo-
somes. Some of the most prominent peaks in the
simulation trajectory of the NUC structure can be assigned
to specific intermediates. The peaks at an extension of
20 nm, 37 nm, or 54 nm clearly originated from breaking
DNA-histone contacts. The subsequent relaxation event at
the 57–66 nm extension reflected the rotation of the protein
core. The positive slope at 66–78 nm extension was due to
the breaking of interactions between histone-dimers. The
large force increase at extension ~85 nm resulted from
numerous single disruption events that arose from a collapse
of secondary structure interactions between two H2A
histone and the N-terminal a-helices of one H2B histone.
The final binding site opening event within the outer turn
in the simulations correlated with the first disruption event
observed experimentally (23) (see Fig. S10).
An unexpected contribution to the force extension curve
was the persistence of histone tail contacts with the inner
DNA turn throughout the stretching simulations. These
induced a partial resolution of secondary structure and
opening of the histone octamer complex. Because this
process was overlaid with breaking of DNA-histone con-
tacts, the identification of distinct intermediates of the un-
wrapping process becomes increasingly difficult (see
Fig. S10). This contribution has, so far, not been considered
in the interpretation of force spectroscopy experiments that
do not provide sufficient resolution to dissect structural
transformations on the nanometer lengthscale. In the virtual
force spectroscopy curve of the NUCDtail structure, the
peaks that preceded the breakage of DNA-histone interac-
tions were less pronounced. The unwrapping of the outer
DNA turn occurred significantly faster, consistent with the
lacking contributions from the H3 N-terminus and the
H2A C-terminus. A corresponding effect was observed at
the 540 bp energy barrier that was largely reduced in the
absence of histone tails.
The investigation of biologically relevant large-scale
motions of the nucleosome is a challenging task because
molecular dynamics simulations are currently limited to
the nanosecond timescale (26,28,29). In contrast, experi-
mental data suggest that processes such as nucleosome
breathing, linker DNA opening, and sliding of nucleosomes
occur spontaneously at the microsecond timescale and
beyond (13,14). One approach to close this gap between
experiments and theory is the use of coarse-grained models
(33,41,42). However, these require some a priori knowledge
of the system features to devise a description that preserves
the characteristic properties to be studied. Alternatively, the
2040 6080 100
I II IIIIVV
8.4 ns 11.8 ns14.0 ns
the SMD simulations, force spectroscopy curves were computed. The
assignment of the five unwrapping phases and intermediates according to
the indicated simulation times corresponds to that given in Fig. 2. The
complete nucleosome structure (red line) is shown in comparison to the
NUCDtail structure (black line).
Virtual force spectroscopy of nucleosome unwrapping. From
Biophysical Journal 101(8) 1999–2008
2004 Ettig et al.
technique of steered molecular dynamics can be applied to
direct motions via an externally applied force, so that the
associated conformational rearrangements can be investi-
gated within a time period that is accessible to MD
So far, SMD has been used primarily to visualize force-
induced protein unfolding events of small systems with
roughly 200 amino acids (29,43–45). Larger systems inves-
tigated with SMD approaches focused mostly on local struc-
tural changes by translocating a relatively small structure
with respect to a bigger macromolecule (46–49). For ex-
was pulled to move through the ribosome. It is noted that the
magnitude and fluctuations of the forces applied in our
constant-velocity SMD simulations depended on the param-
cussed previously, a limitation of the SMD simulations is it
that the currently available computational resources impose
the use of stretching velocities that are orders-of-magnitude
faster than those used in the experimentally conducted force
spectroscopy experiments (29,36–38). Accordingly, it is ex-
pected that random structural fluctuations on a larger time-
scale than that observed here could largely facilitate the
unwrapping process at lower stretching speed. Furthermore,
force peak values larger than those recorded in experiments,
and a loss of details on conformational changes that occur
during the force-induced transitions, are to be expected.
Nevertheless, the currently available cases of SMD simula-
tions in comparison with experimental data demonstrate
that valuable information can be obtained that was later vali-
dated in experimental studies (29).
Here, we applied a combination of MD and SMD to
dissect the details of DNA-histone protein interactions,
and to investigate the process of unwrapping the DNA
from the histone octamer core. Our MD and SMD simula-
tions revealed nucleosome dynamics for all-atom structures
to identify the biologically relevant structural transitions at
a resolution that cannot be reached in the experimental
studies. The molecular dynamics of the nucleosome in an
approximately physiological aqueous environment provided
a DNA-histone interaction map at bp resolution (Fig. 1 A
and see Fig. S2). From the analysis, a five-nucleotide
periodicity pattern emerged that originated from contacts
alternating between the two DNA strands of the double-
helix. This is in agreement with conclusions from recent
force spectroscopy experiments (22). Previously published
histone-DNA interaction maps based on the crystal structure
of the nucleosome (1,2) or on stretching experiments
(22,23,50) identified a ~10-bp periodicity of interactions
of the DNA with the histone proteins. These were assigned
to 14 main interaction sites at regions where the minor
groove faces inwards. Our MD analysis indicates that each
of these interaction sites can be considered as comprising
two ~5-bp separated contacts between each of the two indi-
vidual DNA strands and the histone octamer.
The main origin of variations between different MD
trajectories arose from the interactions of the histone tails
with the DNA. Due to their flexibility they can bind to
DNA in numerous conformations. This is reflected by the
lack of defined structural information on their location in
the available crystal structures. In addition, it is clear from
the asymmetric and different location of the tails observed
in our simulations that various positions are equally
possible. At this point of time, we are unable to evaluate
the effect of the conformational flexibility of the tails in
the simulations in a systematic manner due to the high
demands in computation time.
Despite these limitations, two important conclusions can
be drawn from the MD simulations: Except for the reloca-
tion of the H2A C-terminus (see Fig. S4) the histone
N-terminal tails are relatively stably attached to a certain
region of the DNA in the 100-ns time regime. Furthermore,
on this timescale, the degree of spontaneous disruption of
DNA-histone interactions at the entry-exit site of the DNA
in the nucleosome as well as at the start/end of the looped
region in the NUC and NUC_loop structures was limited
to 1–2 bp. This is in agreement with experimental studies,
concluding that this almost completely wrapped state has
a lifetime of several seconds that are interrupted by open
periods of a few tenths of a second, in which up to 80 bp
of nucleosomal DNA are spontaneously exposed (13–15).
For this process, the energy barrier at 570 bp, which is
stabilized by interactions of the H3 tail and the C-terminus
of H2A, has to be broken.
The comparative SMD analysis conducted here revealed
a large influence of the unstructured histone tails on the
stability and mobility of nucleosomes (Figs. 2–4 and see
Movie S2 and Movie S3). This is supported by previous
MD simulations of nucleosome dynamics (28) and the
MD simulations presented here. Our SMD analysis is in
good agreement with force spectroscopy experiments (21).
These show that the amount of outer-turn DNA wrapping
was reduced by ~60% if the histone tails were removed.
The primary contribution to this effect was from the H3
and H4 N-terminal tails. This agrees with our conclusion
on the H3 tail interactions as opposing unwrapping of outer
turn DNA. In addition, the removal of the H2A/H2B tails
reduced histone-DNA interactions at ~536 bp in the exper-
iments, which is in line with our findings on the contribution
of the H2A tail in the SMD simulations. In the cell, the H3
and H4 interactions are subject to regulation via acetylation
or methylation of lysine residues.
In the in vitro experiments, the acetylation of histones H3
and H4 is clearly apparent in the experimental force spec-
troscopy curves as a factor that weakens histone-DNA inter-
actions (21). Furthermore, numerous proteins recognize
specifically modified histone tails as binding signals and
would disrupt the tail-DNA interactions in their bound state.
These include, for example, heterochromatin protein HP1
binding the H3 tail via its chromodomain (51), the
Biophysical Journal 101(8) 1999–2008
Nucleosome Unwrapping Simulation2005
interactions of the PCAF, Brd2, Brd4, and BRDT bromodo-
mains with acetylated H4 tails (52), and the binding of the
H2A C-terminus to linker histone H1 (5). Our SMD studies
revealed large contributions from the N-termini of H2A,
H2B, H3, and H4 and the C-terminal histone H2A tail to
DNA binding within the nucleosome. In particular, the
N-terminus of H3 and the C-terminus of H2A counteracted
the initiation of DNA unwrapping by stabilizing DNA inter-
actions at positions ~570 bp (Figs. 2 and 3 A). Binding of
these tails by the protein factors mentioned above would
prevent their DNA association and could represent a mecha-
nism to facilitate unwrapping of a certain nucleosome. The
second barrier opposing unwrapping of nucleosomal DNA
was mapped in our simulations to the 545 bp positions.
These two regions were identified also in experimental
studies as sites of strong DNA-histone interactions (22,23).
A direct comparison of the simulated force-extension curves
with the data from Mihardja et al. (23) is shown in Fig. S10
and depicts two distinct transitions in both the simulated
and experimental curves: The removal of the outer DNA
has been referred to as the ‘‘unwrapping event of the outer
DNA turn’’ in the experimental analysis (23), and a good
agreement between the two curves is apparent. A second
significant decrease of force was observed in our analysis
that was assigned to the ‘‘unwrapping event of the inner
DNA turn’’ in the experiments (23). However, although this
part of nucleosomal DNA dissociation occurred at about
the same DNA extension in both the experiments and
simulations, itdidnotrepresentadefined unwrappingtransi-
tion in the simulations. The SMD trajectories revealed
numerous local dissociation events, DNA bending, and
conformational rearrangements that took place over a rela-
tively large force regime as opposed to a sudden release of
the outer DNA turn. In general, an unwrapping step might
not be apparent in the experimental force-extension curves
due to the complex superposition of several processes: 1),
linker DNA bends into the direction of the applied force,
2), DNA stretches, 3), DNA-histone contacts open, 4), the
histone octamer rotates, and 5), its secondary and tertiary
ment of peaks to a single conformational transition in the
experimental force-extension curve is fraught with difficul-
ties. In contrast, the trajectory of the SMD simulations re-
vealed all details of the unwrapping process. From the
complex response of the nucleosome conformation to the
applied force, we identified a sequence of five main phases
of the unwrapping process that are depicted in Fig. 2: Phase
1), Bending of linker DNA. Phase 2), Unwrapping of the
outer DNA turn. Phase 3), Protein core rotation. Phase 4),
ture if histone tails are present. Phase 5), The unwrapping of
the inner DNA turn. It is noted that within each of these five
erable so that these regimes partly overlap.
Our MD and SMD simulations elucidate how histone-
DNA interactions determine access to the DNA sequence
information. The results are relevant in the context of
modeling competitive transcription factor binding and the
conformational properties of the nucleosome chain. The
energy potential of the DNA unwrapping process was
described in a previous study by a spool (representing the
histone octamer) from which the adhesive DNA tube was
removed when tension was applied (39,40). This coarse-
grained model explained several features of the unwrapping
process, and was applied to improve force spectroscopy
simulations of nucleosome chains (53). However, it lacks
details on the shape of the nonhomogeneous histone-DNA
potential along the nucleosome surface revealed here. In
particular, a histone tail-dependent widening of the histone
core during the unwrapping process has not been considered
previously. This opening occurred mostly via conforma-
tional changes in the two H2A$H2B dimers, which is
consistent with previous experimental findings. In these
a hexasome particle was identified as an intermediate during
nucleosome (dis)assembly that lacked one H2A$H2B dimer
(54). Thus, the H2A$H2B dimers appear to be the least
stable part of the nucleosome. Their conformational flexi-
bility could make a significant contribution to the histone-
DNA interaction dynamics.
In summary, the MD and SMD simulations conducted
here identify several features of the mechanism by which
DNA unwraps from the histone octamer core that need to
be considered in coarse-grained descriptions of the nucleo-
some. This leads to integrative modeling approaches as for
of transcription factors to nucleosomal DNA (11). Further-
more, it is anticipated that SMD simulations of the nucleo-
some along the lines described here will provide insights
into the molecular mechanisms by which chromatin remod-
eling complexes translocate nucleosomes along the DNA.
Additional methods, results, 10 figures, and three movies are available at
We are grateful to Karin Voltz for coordinates of an equilibrated nucleo-
some structure, Robert Scho ¨pflin for critical reading of the manuscript,
and Marc Kirchner and Stefan Fischer for valuable discussions.
This work was supported by Deutsche Forschungsgemeinschaft grant
Ri 1283/8-1 and within project EpiGenSys by the Federal Ministry of
Education and Research (BMBF), partners of the ERASysBioþ initiative
supported under the European Union ERA-NET Plus scheme in FP7.
Computations were conducted within project mvb00007 of the North
German Supercomputing Alliance (HLRN) and BMBF grant 01IG07015G
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