Extended Data Figure 4: Analyses of ISWI regulation by AutoN. | Nature

Extended Data Figure 4: Analyses of ISWI regulation by AutoN.

From: Structure and regulation of the chromatin remodeller ISWI

Extended Data Figure 4

a, Representative curves of the MESG-based assays to measure the ATPase activities of MtISWI (81–723) with intact interface (Core; black), R149A/R151A (2RA; blue), and R141A/R149A/R151A (3RA; red). The assays were performed in the absence (open circle) and presence (filled circle) of DNA. The rates of ATP hydrolysis were extracted from the slops of the curves in the linear ranges. The activities were normalized to the ATPase activity of the Core protein in the presence of DNA. The right panel shows the quantification of the measurements in the presence (filled bars) and absence (open bars) of DNA. Error bars, s.d. (n = 3). b, Gels of the restriction-enzyme-accessibility assays of MtISWI (81–723) with the intact interface (Core) and five L3 loop mutants. The assays were performed with 3 nM Cy5-labelled mononucleosomes, and 5 μM of various ISWI proteins at the indicated time points. Owing to the very low activity of the enzymes, a large excess of the proteins was used. c, Quantification of the remodelling activities in b. Core, black; R151A, green; R149A, cyan; R141A, brown; 2RA, blue; 3RA, red. Error bars, s.d. (n = 3). d, Gels of the restriction-enzyme-accessibility assays of MtISWI (81–723) with the intact interface (Core), ΔL3 loop, and Δα4 mutants. Quantification of the remodelling activities is showed in Fig. 2f. The assays were performed with 3 nM mononucleosomes and 0.2 μM of various ISWI proteins. e, Gels of the restriction-enzyme-accessibility assays of core2 mutants of MtISWI (81–723). The cut fractions were quantified and shown in Fig. 2i. Three independent assays were performed and one is shown.

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