FIGURE 3.
Increased efficiency of remodeling H2A.Z-containing nucleosomes is a general feature of ISWI family remodeling enzymes. A, colloidal blue staining of purified remodeling enzymes. B, sample time courses of restriction enzyme accessibility remodeling reactions for core motor proteins on P32 end-labeled nucleosome substrates. C, graphs of phosphorimaging quantitation of uncut substrate versus time from reactions shown in B. kobs for remodeling reactions is determined for the enzyme indicated by fitting the reaction to the exponential decay curve [S] = [S]ie−kt where [S]i is the initial substrate concentration and k is kobs. The ratios of kobs are shown on the right. D, summary of ratio kobs for reactions performed with recombinant H2A.Z-containing nucleosomes to recombinant canonical nucleosomes. The error bars represent standard deviations from the average of 7–26 different remodeling reactions from multiple enzyme preparations and nucleosome assemblies. E, kobs was measured by the rate of appearance of P32 labeled phosphate and is displayed as the ratio between H2A.Z nucleosomes to canonical nucleosomes. Free phosphate was resolved from hydrolyzed ATP by thin layer chromatography in 1 m formic acid, 0.5 m LiCl. F, rates were calculated and graphed as the percentage of the maximal rate within each titration to compare between replicates. The error bars represent the standard deviation between three replicates.