Mechanism of chromatin remodeling
Yahli Lorch,1, Barbara Maier-Davis, and Roger D. Kornberg
Department of Structural Biology, Stanford School of Medicine, Stanford, CA 94305
Contributed by Roger D. Kornberg, January 12, 2010 (sent for review December 16, 2009)
Results from biochemical and structural studies of the RSC
chromatin-remodeling complex prompt a proposal for the remodel-
ing mechanism: RSC binding to the nucleosome releases the DNA
from the histone surface and initiates DNA translocation (through
one or a small number of DNA base pairs); ATP binding completes
translocation, and ATP hydrolysis resets the system. Binding energy
thus plays a central role in the remodeling process. RSC may disrupt
histone-DNA contacts by affecting histone octamer conformation
and through extensive interaction with the DNA. Bulging of the
DNA from the octamer surface is possible, and twisting is unavoid-
able, but neither is the basis of remodeling.
histones ∣ nucleosomes ∣ RSC ∣ DNase ∣ exonuclease III
N ucleosomes inhibit transcription, DNA repair, and other
chromosome transactions. SWI/SNF and RSC chromatin-
remodeling complexes relieve this inhibition by sliding or dis-
assembling nucleosomes (1–4). In the case of sliding, the struc-
ture of the nucleosome is unaltered at the end of the reaction.
Studies to date have revealed an important principle of the re-
modeling process, DNA translocation driven by ATP hydrolysis,
but the underlying mechanism remains obscure. Translocation
slides the DNA around the histone octamer (5–7). Sliding ex-
poses DNA at one end of an isolated nucleosome particle and
in the linker regions between nucleosomes in an array. Exposure
of the DNA may be detected by an increase in susceptibility to
attack by nucleases.
Translocation is effected by the Sth1 subunit of RSC, a mem-
ber of the DEAD/H-box family of helicase/translocases. These
enzymes contact DNA through two domains and step along one
strand by a scissors-like motion between them (8). A gap in one
strand stops this stepping and blocks translocation. It could be
inferred from the effect of gaps in nucleosomal DNA that Sth1
contacts one strand about two turns from the dyad of the nucleo-
some (6). A favored notion for remodeling is that the translocase
draws DNA into the nucleosome from one side, creating a bulge,
which is expelled on the other side. The alternative of DNA twist-
ing strain rather than a bulge traversing the nucleosome has been
excluded on the basis of studies with nicked or gapped nucleoso-
mal DNA (6, 9).
An outstanding question for remodeling by DNA translocation
is how the remodeler effects DNA sliding in the face of histone–
DNA interaction. Translocation by Sth1 entails DNA sliding
from the end of the nucleosome to a point near the dyad, requir-
ing the disruption of all histone–DNA contacts along the way.
Neither the mechanism nor the energetics of this process has
been described.
Here we report on the nuclease digestion of RSC–nucleosome
complexes, alone and in the presence of nucleotides. The results
are supportive of structural studies showing a perturbation of nu-
cleosome structure in the presence of RSC alone (10). These and
other findings lead to a proposal for the mechanism of chromatin
remodeling by RSC.
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Results
DNase I Digestion of a RSC-Nucleosome Complex. DNase I digestion
of the nucleosome has been most informative about histone–
DNA interaction in the past. Digestion of end-labeled nucleoso-
mal DNA and gel electrophoresis produces a pattern of bands
3458–3462 ∣ PNAS ∣ February 23, 2010 ∣ vol. 107 ∣ no. 8
with the periodicity of the DNA double helix, due to exposure
of the DNA to solution alternating with protection by the histone
surface (Fig. 1A, lane 3). As previously reported (11), the band
pattern obtained from a RSC–nucleosome complex is also modu-
lated with the periodicity of the double helix (Fig. 1A, lane 4). It
has been noted, however, that RSC has a general protective effect
(6, 12), consistent with the proposal from structural studies for
binding of the nucleosome in a central cavity. This general pro-
tection is evidenced by a 3.2-fold greater amount of uncut nucleo-
somal DNA in the presence of RSC at the level of digestion used
here (Fig. 1A, lanes 3 and 4). RSC-binding also results in a
hypersensitive site two turns from the dyad [Fig. 1A (arrow),
and C], attributed to interaction with Sth1 (6).
Close examination reveals further differences between the
DNase I digestion patterns of a RSC–nucleosome complex and
the nucleosome alone: RSC-binding results in increased exposure
of sites in periodically protected regions. Indeed, RSC exposes all
major cutting sites in naked DNA within protected regions up to
100 bp from the labeled end of the nucleosome [Fig. 1A and B
(arrows)]. A similar result was obtained upon mapping from the
opposite end of the nucleosome: RSC binding exposed all major
DNA cutting sites in protected regions up to 90 bp from the la-
beled end in the nucleosome. Thus RSC binding even in the ab-
sence of ATP diminishes the interaction of the histones with
almost the entirety of the nucleosomal DNA.
RSC was shown previously to bind naked DNA as tightly as a
nucleosome (13). We now find, by gel shift analysis, that the af-
finity of RSC for DNA is approximately twofold greater for
160 bp DNA than for 64 bp. RSC can evidently accommodate
the full length of nucleosomal DNA.
DNase I Digestion of a RSC-Nucleosome Complex in the Presence of
Nucleotides. As previously noted (11), a RSC–nucleosome com-
plex in the presence of ATP gave a DNase I digestion pattern
closely similar to that of naked DNA (Fig. 1A, lane 5). The
hypersensitive site was abolished. Prior destruction of the ATP
by treatment with hexokinase and glucose produced a digestion
pattern similar to that of the RSC–nucleosome complex in the
absence of ATP (Fig. 1A, lane 6). Once attributed to an alteration
of nucleosome structure by RSC and ATP, this result can now be
understood in terms of sliding of the DNA and its exposure in
truly naked form beyond the ends of the nucleosome.
Addition of the “nonhydrolyzable” ATP analog ATPγS to a
RSC–nucleosome complex enhanced DNase I cutting in pro-
tected regions and, like ATP, abolished the hypersensitive site
(Fig. 1A, lane 7). As little as 15 sec exposure to ATPγS was
sufficient to obtain this result. Pretreatment with a vast excess
of hexokinase and glucose led to the reappearance of the hyper-
sensitive site (Fig. 1A, lane 8), indicative of the destruction of
ATPγS, due to a low activity of kinase on the analog, as has been
observed with other ATPases and phosphotransferases.
Author contributions: Y.L. and R.D.K. designed research; Y.L. and B.M.-D. performed
research; Y.L. and R.D.K. analyzed data; Y.L. and R.D.K. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: lorch@stanford.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
1000398107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1000398107
 Fig. 1. Effects of RSC- and ATPγS binding upon DNase I digestion of nucleosomal DNA. (A) DNase I digestion patterns of naked DNA and nucleosomes, under
the conditions indicated above the lanes. Digestion was with 0.33 units of DNase I (ProMega) for 15 sec (lanes 1 and 2) or with 0.6 units for 40 sec (lanes 3–8).
Positions of size markers produced by digestion of naked DNA with either Msp I or Alu I are indicated by DNA length on the right. (B) Profile of radioactivity in
lane 4 of the region indicated by the bracket on the right of (A). Arrows identify cutting sites exposed by RSC–nucleosome interaction. (C) DNase I digestion
pattern in the vicinity of the hypersensitive site indicated by the arrow in (A), under the conditions indicated above the lanes. In this case, the nucleosomal DNA
was labeled at the end opposite to that in (A). Amounts of enzyme and times of digestion were the same as in (A).

It may be asked whether the effects of ATPγS on the RSC– cause the enzyme is specific for duplex DNA, and upon digestion
nucleosome digestion pattern are attributable to a low level of from both 30 ends to the midpoint, no duplex remains. In the pres-
RSC activity on the analog as well. To investigate this possibility, ence of ATP, digestion proceeds even more rapidly to the mid-

we measured rates of restriction endonuclease cutting of nucleo-
somal DNA. Xmn I, which cleaves a site 20 bp from one end of
the nucleosome, cut about 50% of the DNA in the presence of
ATP in 1 h, but showed no detectable cutting in the presence of
ATPγS or in the absence of any nucleotide (Fig. S1). Msp I, which
cleaves a site 39 bp from the opposite end of the nucleosome, cut
about 70% of the DNA in the presence of ATP in 10 min, but cut
only 10% in 1 h in the presence of ATPγS or in the absence of any
nucleotide (Fig. S1). ATPγS evidently supports little if any RSC
action on the nucleosome.
Exonuclease III Digestion of a RSC-Nucleosome Complex. Exonu-
clease III (exo III), which degrades duplex DNA from the 30
end, is inhibited by histone–DNA interaction, and can be used
to define the boundaries of the core particle of the nucleosome
(14). On more extensive digestion, exo III invades a nucleosome
and produces a pattern of bands differing by multiples of 10 bases
(15) (Fig. 2, lane 1). This pattern, attributed to the dissociation of
DNA–histone contacts occurring every turn of the helix, and to
the advance of exo III upon dissociation, provided evidence of
sequential unraveling of DNA from the ends of the nucleosome.
Exo III digestion reaches a distance 40–50 bp from the ends
before stalling (Fig. 2, lane 1), showing that the DNA associated
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BIOCHEMISTRY
point, as expected from sliding of the nucleosome and exposure
of the DNA (Fig. 2, lane 3). Digestion in the presence of ATPγS
was indistinguishable from that with RSC alone (Fig. 2, lane 4).
Reversible Dissociation of H2A-H2B Dimers. As mentioned above,
one H2A-H2B dimer is undetectable in the structure of a RSC–
nucleosome complex (10). It has long been known that histones
are lost from chromatin in solutions of very low ionic strength in
the presence of naked DNA (16). We observed the loss of his-
tones from an isolated nucleosome in a solution of 15 mM Hepes,
3 mM MgCl2 , in the presence of a 1,000-fold excess of nonspecific
DNA, as shown by an electrophoretic mobility shift of the nucleo-
some (Fig. 3 Center). The addition of as little as 5 mM potassium
acetate prevented this loss of histones (left half of Fig. 3). The
process required nonspecific DNA as a histone acceptor and
was entirely reversible: Addition of potassium acetate following
histone loss restored the particle to the original mobility (right
half of Fig. 3). The histone-depleted particle was indistinguish-
able in mobility from a complex of the H3-H4 tetramer and
DNA. We conclude that H2A-H2B dimers are in association
equilibrium with the tetramer particle of the nucleosome.
We investigated the specificity of the potassium acetate effect.
Substitution of sodium or ammonium for potassium made no dif-

with H2A-H2B dimers is more accessible than that associated
with the central H3-H4 tetramer. In the presence of RSC, diges-
tion proceeds even further, to about half the length of the starting
DNA [Fig. 2, lane 2 (asterisk); digestion further than seen in
ref. 13, due to the use of a three- to fivefold greater excess of
RSC]. Exo III digestion cannot proceed beyond this length be-
ference, showing the cation was not responsible for the effect.
The nature of the anion was important, however, as the midpoint
of the tetramer particle—nucleosome transition shifted from
3 mM for acetate to 5–10 mM for formate, glutamate, and
phosphate, whereas chloride failed to protect the nucleosome
against histone loss at any concentration tested.

Lorch et al. PNAS ∣ February 23, 2010 ∣ vol. 107 ∣ no. 8 ∣ 3459

Fig. 2. Effect of RSC-binding upon exo III digestion of nucleosomal DNA.
Digestion of nucleosomes (2.5 ng DNA) was performed with 40 u exo III
(New England Biolabs) for 5 min under the conditions indicated above the
lanes (0.3 μg RSC, 0.5 mM ATP or ATPγS). Position of size marker produced
by digestion of naked DNA with Msp I is indicated by DNA length on the
right. Product of limit exo III digestion, approximately half the length of
the starting nucleosomal DNA, is indicated by an asterisk.
Discussion
RSC-Binding Reduces DNA–Histone Interaction. Remarkably, RSC
binding alone, without ATP, exposes nucleosomal DNA to attack
by nucleases. In the presence of RSC, DNase I cuts the underside
Fig. 3. Dissociation of H2A-H2B dimers from nucleosomes and reversal in
the presence of acetate. Nucleosomes (1 ng DNA) were incubated with naked
Downloaded at Pakistan: PNAS Sponsored on January 9, 2020
DNA (1 μg pUC19) for 5 min and analyzed by gel electrophoresis. For lanes to
the left of the center line, incubation was in the presence of potassium acet-
ate at the concentrations indicated; for lanes to the right of the center line,
incubation was with naked DNA alone, followed by the addition of potas-
sium acetate at the concentrations indicated. The bands in the gel correspond
to the nucleosome and tetramer particle. The relative intensities are plotted
as black diamonds and red squares below.
3460 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1000398107
of the double helix, normally protected by interaction with the
histone octamer of the nucleosome; and exo III invades DNA
normally protected by interaction with the central H3-H4 tetra-
mer, digesting all the way to the dyad of the nucleosome.
These results are consistent with cryoelectron microscopy
(cryoEM) and 3D reconstruction of the RSC–nucleosome
complex (10). Whereas density for all the histones except one
H2A-H2B dimer could be seen in the reconstruction, density
for much of the DNA, including that near the ends (entry/exit
to the nucleosome) and near the dyad, was conspicuously absent.
The perturbation of histone–DNA interaction revealed by nucle-
ase digestion may be manifest in the cryoEM reconstruction as a
loss of DNA density due to motion or disorder.
The surprising disappearance of an H2A-H2B dimer from the
cryoEM reconstruction also receives support from our work. We
find that H2A-H2B dimers are reversibly dissociable from the
nucleosome, in a manner that is critically dependent on ionic con-
ditions. Perturbation of histone-DNA interaction by RSC-binding
may promote the same dimer dissociation as occurs in the ab-
sence of RSC under appropriate ionic conditions. Binding in
the RSC cavity may even create such ionic conditions.
Biochemical and structural studies thus converge on the con-
sequences of RSC–nucleosome interaction. The energy of the
interaction is sufficient to disrupt histone-DNA contacts and re-
lease the DNA from the histone octamer surface. The DNA is
free to slide as required for translocation.
RSC-Binding Promotes DNA Exposure near the Dyad of the
Nucleosome. The most prominent feature of the DNase I digestion
pattern of the RSC–nucleosome complex is a hypersensitive site
about two turns from the dyad. Hypersensitivity is abolished by
the addition of ATPγS. Two observations indicate this effect is not
due to hydrolysis of ATPγS. First, the DNase I digestion pattern
in the presence of ATPγS is otherwise the same as that obtained
with RSC alone, in contrast with that in the presence of ATP.
Second, sliding through many base pairs, evidenced by exposure
of restriction endonuclease sites, and which requires ATP hydro-
lysis, does not occur in the presence of ATPγS. Hypersensitivity is
most simply explained by strain on the DNA, perturbing the dou-
ble helical structure or enhancing its displacement from the
histone octamer surface. Strain may be caused when the translo-
case engages the nucleosomal DNA, initiating DNA transloca-
tion, and be relieved when ATP binding completes the translo-
cation process.
Proposed Remodeling Mechanism. The prevailing view of chromatin
remodeling by RSC and the related SWI/SNF complex starts
from the lack of effect of nicks or gaps on the process. Remodel-
ing occurs up to the point where the translocase encounters a
strand break (6, 7). On the basis of these findings, torsional strain,
resulting from screw rotation of the DNA, has been ruled out as a
driving force for the process (6, 9). In the absence of screw rota-
tion, translocation of the DNA can only occur by sliding. As the
structure of the nucleosome is conserved during remodeling, the
double helix must remain in register with interaction sites on the
histone surface, so sliding must occur through one or more turns
of the double helix. Action of the translocase, drawing in a turn of
the helix, would create a small bulge protruding from the histone
surface, thought to diffuse to the opposite end of the nucleosome
in the manner of a wave traversing the particle.
There are several limitations to this line of reasoning. First, the
lack of effect of nicks and gaps does not exclude a mechanism
involving screw rotation of the DNA. It only shows that torsional
strain is not required for the process. Second, sliding through a
turn of the double helix is unlikely, because translocases closely
related to Sth1 move one base step at a time. Finally, a small
bulge of a turn or two of the double helix is energetically disfa-
vored by the low bending flexibility of DNA.
Lorch et al.

Fig. 4. Proposed mechanism of chromatin remodeling. Schematic of
nucleosome at left depicts histone octamer as gray disk and DNA as solid
curve. Schematic of RSC–nucleosome complex in center depicts RSC as yellow
volume surrounding the nucleosome, with Sth1 region in red, with positively
charged surface for interaction with the nucleosome, and with DNA
largely dissociated from surface of the histone octamer. Schematic of RSC–
nucleosome at right shows DNA translocated in the presence of ATP.
The fundamental problem confronting any proposed mecha-
nism of remodeling is the extensive histone–DNA interaction
in the nucleosome. The histones bind through an intricate pattern
of dipolar, nonpolar, hydrogen bonding, and electrostatic inter-
actions to both strands of the DNA every turn of the helix (17).
Simple rotation or sliding, requiring the simultaneous rupture of
all contacts, is evidently impossible. Mechanisms involving tor-
sional strain or DNA bulge formation solve the problem through
a local perturbation of the double helix that diffuses along the
nucleosomal DNA, disrupting only one or a small number of
histone–DNA interactions at a time. Neither mechanism, how-
ever, addresses the central question of how the translocase draws
in DNA, whether for strain or bulge formation, in the first place.
The translocase, located two turns from the dyad of the nucleo-
some, must draw in DNA from the linker five turns away,
requiring the disruption of dozens of histone–DNA contacts
along the way. This problem arises at every step of the translocase
on the DNA.
It may be asked whether the spontaneous unwrapping of nu-
cleosomal DNA, well characterized by many procedures, might
relieve the inhibitory effect of histone–DNA interaction upon
translocase activity. The answer is that the rate of spontaneous
unwrapping is far too slow. Unwrapping an end of the nucleoso-
mal DNA occurs on the order of a second (18); the equilibrium
constant for unwrapping to the middle of the nucleosome is at
least 103 -fold less than that for an end (19, 20), so the frequency
of unwrapping to the middle is on the order of one per hour (on
the reasonable assumption that rewrapping is not slower in the
middle than at an end).
The solution of the problem is unwrapping upon RSC bind-
ing to the nucleosome (Fig. 4). Both cryoEM and nuclease diges-
tion provide clear evidence of unwrapping all the way to the dyad
of the nucleosome. The DNA is substantially free of the histones
and available for translocation. A nuclease hypersensitive site
appears at the location of Sth1 due to engagement of the active
center, straining the DNA toward translocation. ATP-binding
results in movement, relieving the strain. ATP hydrolysis resets
the system.
Movement of the DNA must occur by screw rotation, in view of
the structural conservation of Sth1 with DEDxx helicases and du-
plex DNA translocases (8). X-ray studies of helicases make a
compelling case for stepping along a single DNA strand, driven
by ATP binding (8). The x-ray structure of Rad54 (21), a translo-
case closely related to Sth1, shows binding to one strand of duplex
DNA nearly identical to that observed for helicases. Rotation of
the DNA due to stepping along one strand imparts approximately
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one superhelical turn for every 10 bases. The resulting torsional
strain need not diffuse past histone–DNA interaction sites nor
will it accumulate and impede translocation because the DNA
is largely free of the histone surface, allowing passage of the strain
to the next linker region. There the strain will be relieved by the
action of untwisting enzymes, abundant in eukaryotic cells, or it
Lorch et al.
will be canceled by translocation in opposite directions by RSC
associated with neighboring particles.
The key to the RSC remodeling mechanism is the role of bind-
ing energy in destabilizing the nucleosome and releasing the
DNA. It may seem paradoxical that RSC protects nucleosomal
DNA from nuclease digestion and at the same time exposes pre-
viously protected sites to nuclease attack. It is this paradox, how-
ever, that explains how RSC binding achieves a destabilizing
effect. RSC contains a deep cavity for binding the nucleosome
(10, 12, 22, 23). It envelops the particle, contacting both DNA
and histones, thereby conferring general protection against nucle-
ase digestion. The cavity is likely positively charged, as RSC binds
naked DNA as tightly as a nucleosome. Moreover, as shown here,
the cavity can accommodate the entire length of nucleosomal
DNA. We may therefore suppose that entry in the cavity facili-
tates unwrapping of the nucleosomal DNA, enhancing the se-
paration of the nucleic acid and protein components of the
nucleosome, while maintaining them in close proximity. The rate
of unwrapping to the middle of the nucleosome would be most
affected because the rate presumably decays exponentially from
the end of the DNA toward the middle. Although positively
charged, the RSC cavity is unlikely to bind DNA so tightly or
in a manner that prevents rotation and consequent relaxation
of torsional strain. A single molecule study of RSC–DNA inter-
action is supportive of this view (24). RSC imparts negative super-
helicity, and so must bind to naked DNA at two sites, one
undoubtedly the translocase active center, and the second pre-
sumably the interior of the RSC cavity. The number of negative
supercoils introduced is, however, far less than one per turn of the
helix translocated, indicative of rotation in the RSC cavity.
RSC–histone interaction may also contribute to destabilization
of the nucleosome. The cryoEM studies (10) suggest RSC inter-
action with the histones, primarily H2A and H2B, on the flat
faces of the nucleosome. RSC may perturb the structure of the
histone octamer, for example, by extending the histone superhelix
(increasing the pitch), causing a reduction in diameter and
release of the DNA. For example, a 1 Å change in diameter, suf-
ficient to perturb histone-DNA interaction, would correspond to
a 1 bp change in contour length of the DNA. The displacement of
BIOCHEMISTRY
an H2A-H2B dimer in the structure of a RSC–nucleosome com-
plex (10) directly demonstrates a perturbation of octamer struc-
ture, though a small change in octamer diameter would not have
been detectable at the resolution of the analysis.
Unwrapping of nucleosomal DNA and release of a dimer,
which enable DNA translocation, are natural processes, intrinsic
to the nucleosome. The binding of H2A-H2B dimers is delicately
balanced against the bending strain imparted to nucleosomal
DNA. The balance is critically dependent on counterion compo-
sition and concentration, and is tipped toward dissociation by the
removal of carboxylate ions that contribute little to the overall
ionic strength. RSC-binding similarly tips the balance, increasing
the rates of unwrapping and of dimer dissociation, while con-
serving the structure of the nucleosome upon its release. It is
the extended lifetime of the unwrapped, partially dissociated
state that results in the apparent loss of DNA and of a dimer from
the cryoEM structure, as well as in the greater accessibility to nu-
clease attack. The unwrapped DNA may remain associated with
the dimer in the RSC cavity, because exo III digestion still pauses
every 10 bases from the end of the nucleosome (Fig. 2).
The natural propensity for unwrapping may also explain why
Sth1 alone is capable of chromatin remodeling (6), although an
order of magnitude more slowly than the entire RSC complex.
Sth1 alone may stimulate the rates of unwrapping and dimer
dissociation, but to a lesser extent than RSC. Sth1 can then trans-
locate nucleosomal DNA, but at a lower rate than RSC (6).
Other chromatin-remodeling complexes may also stimulate
the natural processes of unwrapping and dimer dissociation.
Many remodeling complexes contain members of the DEDxx
PNAS ∣ February 23, 2010 ∣ vol. 107 ∣ no. 8 ∣ 3461
 helicase/translocase family and may be presumed to function by
DNA translocation (6, 25), thus depending on nucleosomal DNA
unwrapping. The Drosophila ACT complex has been reported to
expose DNA near the entry and exit points of the nucleosome
(26). The SWR1 complex (27), whose ATPase subunit is related
to Sth1, catalyzes the exchange of H2A-H2B dimers for Htz1-
H2B dimers, presumably by promoting the intrinsic process of
dimer dissociation.
Materials and Methods
Nucleosomes were prepared with the use of 160 bp DNA 32 P-labeled at one 50
end (by PCR with end-labeled primer) and rat liver histones (9, 28). RSC was
prepared as described (28). Except for DNase I digestion, all reactions were in
15 mM Hepes, pH 8.0, 3 mM MgCl2 , 100 μg∕mL BSA in a volume of 15 μL at 30
°C. Electrophoresis was in a 7% polyacrylamide—7 M urea gel in Tris/Borate/
EDTA buffer for DNase I and exo III experiments, in a 7% polyacrylamide gel
in Tris/Acetate/EDTA buffer for restriction endonuclease digestion experi-
ments, and in a 3.2% polyacrylamide gel in 10 mM TrisCl, pH 7.5, 1 mM EDTA
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3462 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1000398107
for nucleosome dissociation experiments. DNA was extracted with phenol–
chloroform–isoamyl alcohol except for nucleosome dissociation experiments,
in which reaction mixtures were applied directly to the gel. Data were
obtained with the use of a PhosphorImager and analyzed with ImageQuant.
Prior to DNase I digestion, DNA (1.2 ng), either naked or in nucleosomes,
was incubated for 20 min at 30 °C in 20 mM Hepes, pH 8.0, 5 mM MgCl2 , 7 mM
potassium acetate, 125 μg∕mL BSA, in a volume of 20 μL, containing 0.8 μg
18mer oligonucleotide (with Gcn4-binding sequence, but only important as
carrier DNA for digestion), and where indicated, 0.5 mM ATP, 0.5 mM ATPγS,
and 0.3 μg RSC. Also where indicated, hexokinase (100 μg) and glucose were
added and incubated 20 min at 30 °C (for destruction of ATP or ATPγS) before
the addition of RSC and subsequent incubation for another 20 min. Finally
DNase I was added in 5 μL of 25 mM Hepes, pH 7.5, 5 mM MgCl2 , 3 mM CaCl2 ,
50 mM potassium chloride, 200 μg∕mL BSA. Digestion was stopped by the
addition of 2.5 μL of 100 mM EDTA.
ACKNOWLEDGMENTS. We thank Brad Cairns and Michael Levitt for discussion.
This research was supported by National Institutes of Health Grant GM36659.
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