Fig. 3

Mutations in brace and post brace regions alter remodelling activity. a Alignment of brace and post brace sequences of yeast Chd1 (yChd1), Snf2 (ySnf2), M. thermophila ISWI human CHD4 (hCHD4) and Drosophila Mi-2 (dMi-2). Structural elements are indicated. Positions of H1196Y and L1215P mutations are shown. b ATPase activities in absence (−) and presence (+) of polynucleosomes. Wild-type dMi-2 activity was set to 100%. Error bars represent SEM from three independent experiments. c Electrophoretic mobility shift assays with 150 nM 0–80 mononucleosome and decreasing dMi-2 concentrations (lanes 2, 6, 10: 900 mM; lanes 3, 7, 11: 450 nM; lanes 4, 8, 12: 225 nM; lanes 5, 9, 13: 113 nM). Positions of nucleosome–protein complexes and mononucleosome are indicated. d Restriction enzyme accessibility assays were performed with dMi-2 proteins (115 nM) and body-labelled 0–80 mononucleosomes (20 nM) for 5, 10, 20 and 40 min. Percentage of remodelled nucleosomes is shown (%DNA cut). Error bars represent SEM from three independent experiments. e Nucleosome sliding assays with 0–77 mononucleosomes (150 nM) and decreasing dMi-2 concentrations. Upper panel: lanes 2, 6, 10: 900 nM; lanes 3, 7, 11: 450 nM; lanes 4, 8, 12: 225 nM; lanes 5, 9, 13: 113 nM). Lower panel: lanes 2, 9: 900 nM; lanes 3, 10: 450 nM, lanes 4, 11: 225 nM; lanes 5, 12: 113 nM, lanes 6, 13: 56.5 nM; lanes 7, 14: 28.25 nM; lanes 8, 15: 14.12 nM. Positions of mononucleosomes and free DNA are indicated. Asterisk: dMi-2/mononucleosome complexes forming at high protein concentrations. Both panels are derived from different experiments. Upper panel is part of gel shown in Supplementary Fig. 9. First two panels are reproduced (WT and WT-ATP) in Figs. 2d, 3e and 4d to aid visual comparison with activity of mutants. f Nucleosome sliding assays with 0–77 mononucleosomes (150 nM) and decreasing hCHD4 concentrations. Lanes 2, 10: 900 nM; lanes 3, 11: 450 nM, lanes 4, 12: 225 nM; lanes 5, 13: 113 nM, lanes 6, 14: 56.5 nM; lanes 7, 15: 28.25 nM; lanes 8, 16: 14.12 nM, lanes 9, 17: 7.06 nM