Figure 3.

Identification of crucial determinants for ISWI function in the H4 N-terminus. (A) ATPase activity of ISWI in the presence of the variant nucleosomes shown in Figure 2. ATPase activity is expressed as the percentage of ATP hydrolyzed during the assay. The bars represent the average of three independent experiments, the variability is indicated by the error bars. (B) Nucleosome ‘spacing’ activity of ISWI in the presence of the variant nucleosomes shown in Figure 2. Removal of the amino acids R₁₇H₁₈R₁₉ abolishes the ‘spacing’ capacity of ISWI. The histone octamers were reconstituted into nucleosomes on plasmid DNA by NAP-1 assisted assembly in the presence of ISWI and subjected to MNase digestion. Chromatinized DNA (300 ng) was digested with 0.2 U of MNase for 20, 50 and 110 s. The resulting DNA fragments were purified and visualized by agarose gel electrophoresis and SybrGold“ staining. The marker is a 123 bp DNA ladder.