Extended Data Figure 6: Analyses of the H4-binding surface of ISWI. | Nature

Extended Data Figure 6: Analyses of the H4-binding surface of ISWI.

From: Structure and regulation of the chromatin remodeller ISWI

Extended Data Figure 6

a, GST pull-down assays. GST–H4 pulled down a significant amount of the 2D mutant MtISWI (81–723) (lane 4). Introduction of additional D520A mutation showed a mild defect in H4-binding (lane 7). The mutations of D524A and E474A dramatically reduced the binding (lanes 10 and 13, respectively). b, Gels of the restriction-enzyme-accessibility assays of the H4-binding interface mutants. The assays were performed with 3 nM Cy5-labelled mononucleosomes and 0.2 μM ISWI proteins. The cut fractions were quantified and shown in Fig. 3e. c, ATPase activities of MtISWI (81–723) with the intact interface (Core) and two mutants of the H4-binding surface (E474A and D524A) in the absence (open bars) and the presence (filled bars) of DNA. Error bars, s.d. (n = 3). d, Chromatin remodelling activities of Core (black), E474A (blue) and D524A (red). The bottom panels show the representative gels of the chromatin remodelling assays. The assays were performed with 3 nM Cy5-labelled mononucleosomes and 5 μM ISWI proteins. Error bars, s.d. (n = 3).

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