Fig. 2

ATPase domain mutations disrupt nucleosome remodelling. a ATPase activities of wild-type and mutated dMi-2 proteins were determined in absence (−) and presence (+) of saturating amounts 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 were carried out with 150 nM of 0–80 mononucleosome and decreasing concentrations of dMi-2 proteins as indicated (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). The positions of nucleosome–protein complexes and unbound mononucleosome are indicated by arrows on the left. c Restriction enzyme accessibility assays were performed with wild-type or mutant dMi-2 proteins (115 nM) and body-labelled 0–80 mononucleosomes (20 nM) as indicated on the right. Reactions were incubated for 5, 10, 20 and 40 min. The percentage of remodelled nucleosomes was determined by dividing the fraction of cut DNA by total DNA at each time point (%DNA cut). Error bars represent SEM and are derived from three independent experiments. d Nucleosome sliding assays were carried out with 150 nM of 0–77 mononucleosomes and decreasing concentrations of dMi-2 proteins as indicated (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). The positions centrally and end positioned mononucleosomes and free DNA are indicated on the left. Asterisk denotes the position of dMi-2/mononucleosome complexes that form at high protein concentrations. All five panels are derived from a single experiment (see Supplementary Fig. 9 for the entire gel). Note that we have reproduced the first two panels (WT and WT-ATP) in Figs. 2d, 3e and 4d to aid visual comparison with the activity of mutants analysed in these figures