Figure 5 |. Nucleosome desymmetrization leads to altered ISWI activity.

(a) Kinetics of remodeling as measured by REAA for the indicated acidic patch mutants relative to wild-type. Errors = s.e.m (n=3 independent experiments). H2A-3A: H2A E61A, D90A, E92A. Data are represented as the mean of experimental replicates. (b) Heatmap representation of the kinetic data from panel a showing location of each mutant on the nucleosome surface (pdb, 1KX5). (c) Schematic of the two step-synthesis used to prepare heterotypic nucleosomes: step one, generation of oriented hexasomes (1a) by varying ratio of DNA, H3-H4 tetramer and H2A-H2B dimer. Step 2, formation of the desired heterotypic nucleosomes (2) carrying a fluorescent label (Cy5, red star). (d) Remodeling activity of SNF2h on indicated homotypic and heterotypic nucleosome substrates. SNF2h was incubated with an end-positioned mono-nucleosome containing a 45 bp overhang in the presence of ATP. Aliquots of the reaction mixtures were analyzed at various time-points by EMSA and monitoring the Cy5 label. (n=3 independent experiments). Uncropped figures are shown in Supplementary Fig. 18. (e) Schematic of the FRET-based remodeling assays employed oriented dinucleosomes containing Cy3 and Cy5 dyes and either heterotypic mutant acidic patch in one monomer (dinuc 3a) or the wild-type acidic patch in both (dinuc 3c). (f) Remodeling activity of SNF2h on dinuc 3a, dinuc 3c and a mixture of mononucleosomes as assessed by the FRET assay (n=3 independent experiments). SNF2h was incubated with indicated substrates in the presence of ATP. Change in normalized FRET signal (see Methods) as a function of time as shown for three replicates of each. Yellow dotted line: curve fitting for change in FRET signal of a mixture of pre-ligated mono-nucleosomes (n=2 independent experiments).