FIGURE 4.
ISWI family stimulation is independent of nucleosome position and dependent on the extended acidic patch on H2A.Z-containing nucleosomes. A, native PAGE of eluted glycerol gradient fractions of mononucleosomes recombinantly assembled on the TPT positioning sequence with a 45-bp overhang. Fractions of similar mobility within the native gel were compared as similarly positioned nucleosomes in REA assays with SNF2H. A summary of ratio of kobs between the indicated fractions is shown in the graph on the right. B, kobs of remodeling determined by REA assay are compared between the mutant H2A.Z mononucleosomes (NK) and canonical mononucleosomes for the enzyme indicated. The calculations were done as in Fig. 3D. The dashed line is from the data shown in Fig. 3D and is displayed as a reference point to compare H2A.Z-NK remodeling to wild type H2A.Z remodeling by SNF2H. C, REA assay with Cy5 end-labeled recombinant mononucleosomes (red) mixed with either Cy3 end-labeled H2A.Z nucleosomes or mutant H2A.Z (NK) mononucleosomes (green). Separate SNF2H and ACF remodeling reactions are indicated. The Cy3 label on the H2A.Z nucleosomes appears relatively less bright than the Cy3 label on the NK nucleosomes most likely because of decay of the Cy3 on the H2A.Z nucleosomes that were assembled earlier.