1. SWI/SNF Chromatin Remodeling Requires Changes in DNA Topology
Igor Gavin, Peter J Horn, Craig L Peterson 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1
ySWI/SNF-Dependent Remodeling Requires DNA Folding into a Nucleosome
(A) ySWI/SNF-dependent remodeling of nucleosomal arrays and nonspecific DNA–histone complexes. HincII (500 U/ml) and ySWI/SNF (2 nM) were added to remodeling reactions that contained either a 11-mer nucleosomal array (1 nM) or a nonspecific histone–DNA substrate (1 nM). Reactions were incubated at 37°C for 20 min followed by addition of ATP to half of the reaction mixture. Aliquots were taken at the indicated times, and the reaction was stopped by addition of 5× stop solution (0.5% SDS, 25 mM EDTA, 1 mg/ml proteinase K). Purified DNA was loaded on a 1% agarose gel to separate digestion products from uncut DNA fragments. The amount of radioactivity in each band was quantified, and the percentage of uncut arrays was calculated. The average from at least three experiments is plotted as a function of time. The standard deviation did not exceed 10%.
(B) ySWI/SNF ATPase activity is equally stimulated by DNA, nonspecific histone–DNA complexes, and nucleosomal arrays. ySWI/SNF ATPase reactions were performed at an ATP concentration of 100 μM (Logie and Peterson 1999). Reactions were stopped by spotting onto thin layer chromatography (TLC) paper. Inorganic phosphate was separated from ATP, quantified, and the percentage of hydrolyzed ATP was calculated. The average from at least three experiments was plotted as a function of time. The standard deviation did not exceed 10%.
(C) Competition of ySWI/SNF remodeling activity by nonspecific histone–DNA complexes and nucleosomal arrays. Remodeling reactions contained HincII (500 U/ml), 1.5 nM ySWI/SNF, and 1 nM 32P-labeled 11-mer nucleosomal arrays. Reactions either lacked an unlabelled competitor DNA (closed circles) or contained 5 nM nonspecific histone–DNA complexes (squares) or nucleosomal (open circles) competitor DNAs.
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Changes in DNA Topology Are Required for SWI/SNF Remodeling
(A) A schematic representation of linear and circular trinucleosomal array substrates. Predicted nucleosome positions are shown as ellipses relative to restriction sites. The hatch mark between each nucleosome indicates the position of EcoRI sites.
(B) ySWI/SNF (2 nM) and MspI (2500 U/ml) were incubated in remodeling reactions that contained either the linear or circular trinucleosomal array (1 nM). The fraction of uncut array was quantified with time as described in the legend to Figure 1.
(C) Linear arrays, circular arrays, and DNA equally stimulate ySWI/SNF ATPase activity at similar concentrations. Reactions were carried out at an ATP concentration of 50 μM for 10 min.
(D) Addition of eukaryotic topoisomerases rescue ySWI/SNF remodeling of small chromatin circles. Topoisomerase I (Amersham; 0.3 U/μl) or topoisomerase II (USB; 1 U/μl) were added to remodeling reactions that contained linear or circular trinucleosomal array, ATP, and MspI. Remodeling was initiated by addition of ySWI/SNF at t = 12 min. The fraction of uncut array was quantified with time as described in the legend to Figure 1.
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 4
ySWI/SNF Enhances the Accessibility of Small Circular Arrays in the Absence of Dramatic Histone Octamer Movements
(A) MNase digestion analysis of nucleosome positions within linear trinucleosomal arrays. End-labeled nucleosomal arrays or free DNA were digested with increasing concentrations of MNase (triangles above lanes). DNA was purified and electrophoresed on a 4% acrylamide gel. Derived nucleosome positions are shown by ellipses. Note that in the presence of ySWI/SNF and ATP, the MNase digestion pattern of the linear arrays is similar to but clearly distinct from that of the free, linear DNA. S, linear array substrate; M, 100 bp DNA ladder.
(B) MNase digestion of circular trinucleosomal arrays. Arrays were reconstituted onto DNA circles that contained a 32P label at the point of ligation. Circular arrays were incubated with increasing concentrations of MNase (triangles above lanes), and the purified DNA was cut with AlwI prior to loading on the gel. Cleavage with AlwI generates the unique, end-labeled DNA fragments (see Figure 2A for schematic). Reactions contained ySWI/SNF, topoisomerase II, and ATP where indicated. C, circular; L, linear; N, nicked DNA probe. Asterisks indicate hypersensitive sites on the naked, circular DNA probe. Note that the MNase digestion pattern of the circular arrays is similar both in the presence or absence of ATP. S, circular array substrate; M, 100 bp DNA ladder.
(C) Restriction enzyme accessibility of circular trinucleosomal arrays. Circular arrays were incubated with topoisomerase II, ySWI/SNF, and either MspI (500 U/ml) or EcoRI (500 U/ml). ATP was added where indicated. For reactions that contained apyrase, circular arrays were first incubated with topoisomerase II, ySWI/SNF, and ATP for 5 min, then apyrase (10 U/ml) was added to remove ATP, and this treatment was followed by addition of either MspI or EcoRI. The amount of radioactivity in all topoisomers was quantified, and the percentage of uncut array was calculated. Data shown is representative of three independent experiments. Note that the bulk of the minichromosomes contain an accessible EcoRI site and that ySWI/SNF action does not lead to a significant decrease in EcoRI accessibility.
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 5
ySWI/SNF-Dependent Loss of Supercoiling of Small Circular Arrays Does Not Result from Eviction of Histone Octamers
Analysis of the MspI accessibility of minichromosome topoisomers. Circular trinucleosomal arrays were incubated with topoisomerase II in the absence (lanes 1–3) or presence (lanes 4–7) of MspI and in the presence (lane 1 and lanes 3–7) or absence (lane 2) of ySWI/SNF. All reactions were incubated in the absence of MspI for 5 min at 37°C, and then, where indicated, MspI was added to half of the mixture, and all reactions were incubated for an additional 20 min. For reactions shown in lanes 6 and 7, apyrase (10 U/ml) was added after the initial 5 min incubation prior to MspI addition. DNA was purified and electrophoresed on a 4% acrylamide gel (30:1 acrylamide:bisacrylamide ratio). The migration of topoisomers and linear DNA is shown on the left. Identity of the topoismers was determined by electrophoresis of topoisomer standards isolated by ligating circles in the presence of different concentrations of ethidium bromide. M, 32P-labeled 100 bp DNA molecular weight standard (GIBCO–BRL).
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 3 ySWI/SNF Changes DNA Topology in Small Domains
Nucleosomal arrays were reconstituted on a 3.1 kb plasmid (pCL115) that contains a single 5S rDNA nucleosome-positioning sequence. Reconstitutions were performed with either the circular plasmid or with the linear plasmid precut with ScaI. Remodeling reactions contained ySWI/SNF (2 nM), nucleosomal substrate (1 nM), and HincII (500 U/ml), for which there is a unique site within the 5S rDNA sequence. The fraction of uncut array was quantified with time as described in the legend to Figure 1.
Molecular Cell 2001 7, DOI: ( /S (01) )