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was stimulated by DNA at least 25 bp in length, but was not further stimulated
by the addition of nucleosomes. A turnover rate of approximately 7.5 molecules
ATP per second was established under optimal conditions. 25,48,49 The Sth1
subunit alone was found to have
2.5 times lower ATPase activity than the
intact Rsc2-RSC complex. 49 ATP hydrolysis by RSC/Sth1 is coupled to 3 0 -5 0
translocase activity. This was demonstrated by DNA length-dependent stimu-
lation of ATPase activity, stimulation by DNA minicircles, and by triplex
displacement activity. 49,50
When DNA and nucleosome binding were tested, the RSC complex bound
both with comparable affinity, but the presence of nucleosomes and ATP
caused a shift to a slower migrating form (called an ''activated'' complex, in
which nucleosomal DNA was more susceptible to DNase I). 47 The change in
nuclease cleavage pattern suggested that the RSC complex was capable of
altering nucleosome structure. Another chromatin-remodeling assay follows
the cleavage of a restriction enzyme site that is inaccessible on the surface of a
nucleosome. Incubation with RSC and ATP induced remodeling, allowing the
site to be cut. 25,47,49 Other documented activities of the RSC complex include
the ability to reposition nucleosomes, transfer histone octamers onto naked
DNA, and dismantle nucleosomes in the presence of a histone chaperone. 51-53
Sth1 alone was five- to sixfold less active than the RSC complex in nucle-
osome mobilization assays, showing the importance of other RSC subunits for
maximal RSC complex activity. 49 One of the functions of the auxiliary subunits
may be to increase the affinity for nucleosomes. Nucleosomes containing tetra-
acetylated H3 were remodeled
16-fold faster than unmodified nucleosomes
due to preferential binding of the acetylated nucleosomes. 53 In addition, RSC
has been shown to have increased affinity for nucleosomes acetylated by
NuA4, 53 suggesting a role for the bromodomain containing subunits (Rsc1,
Rsc2, Rsc4, and Sth1) in recruitment to chromatin and maximum remodeling
activity. The majority of these in vitro assays have been performed using either
a mixed population of Rsc1 and Rsc2-containing complexes or only the Rsc2-
containing isoform; one outstanding question is whether the two isoforms
possess identical biochemical activity.
The molecular mechanism of nucleosome remodeling has been examined
using single-molecule experiments. RSC was shown to form relaxed super-
coiled loops of around 400-700 bp in an ATP-dependent manner on DNA
tethered and stretched at low forces with a magnetic trap. 54 These loops could
be visualized using atomic force microscopy. The formation of loops also
occurred on nucleosomal templates, resulting in loops of between 20 and
1200 bp (average of
100 bp), but these loops could form at higher tensions. 55
Slippage of loops was observed on both templates, but the translocation rate
was greater on naked DNA than on nucleosome-bound DNA (
500 bp per s
compared with 12 bp per s). Together, these data suggest that nucleosome
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