Fig. 4

Chromodomains mutations abrogate nucleosome remodelling. a ATPase activities of wild-type and mutated dMi-2 proteins in absence (−) and presence (+) of polynucleosomes. ATPase activity of wild-type dMi-2 was set to 100%. Error bars represent SEM and are derived from three independent experiments. b Electrophoretic mobility shift assays with 150 nM of 0–80 mononucleosome and decreasing dMi-2 concentrations (lanes 2, 6, 10, 14: 900 mM; lanes 3, 7, 11, 15: 450 nM; lanes 4, 8, 12, 16: 225 nM; lanes 5, 9, 13, 17: 113 nM). Positions of nucleosome–protein complexes and mononucleosome are indicated. c Restriction enzyme accessibility assays with wild-type or mutant dMi-2 proteins (115 nM) and body-labelled 0–80 mononucleosomes (20 nM). Reactions were incubated for 5, 10, 20 and 40 min. Percentage of remodelled nucleosomes is shown (%DNA cut). Error bars represent SEM from three independent experiments. d Nucleosome sliding assays with 0–77 mononucleosomes (150 nM) and decreasing dMi-2 concentrations (lanes 2, 6, 10, 14, 18: 900 nM; lanes 3, 7, 11, 15, 19: 450 nM; lanes 4, 8, 12, 16, 20: 225 nM; lanes 5, 9, 13, 17, 21: 113 nM). ATP was omitted from reactions shown in lanes 2 to 5 (−ATP). Positions of mononucleosomes and free DNA are indicated on the left. Asterisk: dMi-2/mononucleosome complexes forming at high protein concentrations. All panels are derived from a single experiment (shown in Supplementary Fig. 9) except C464Y. The first two panels (WT and WT-ATP) are reproduced in Figs. 2d, 3e and 4d to aid visual comparison with the activity of mutants. e Structure of the yCHD1 double chromodomain–ATPase domain fragment (3MWY) (Hauk/Bowman). Inset shows the basic loop of chromodomain 1 of yChd1 (green). The superimposed relative positions of V558 and R572 of CHD4 are shown in yellow. Chromodomain 1: light blue, chromodomain 2: cyan, ATPase domain core 1: orange, ATPase domain core 2: red