A single amino acid substitution within the
ATPase domain of both yeast ISWI proteins inactivates ATP-dependent
activities of the yeast ISWI complexes. (A) Subunit
composition of the mutant yeast ISWI complexes. Purified wild-type and
mutant yeast ISWI complexes were separated on an 8% SDS-polyacrylamide
gel and stained by silver. (Arrows) The subunits of the complexes.
(●) Proteolytic products; (*) contaminants. (B) Mutant
yeast ISWI complexes are defective in the ATPase activities. The ATPase
assays were done in buffer (B) or in the presence of nucleosomes (N).
Hydrolysis/complex per min denotes the molecules of ATP
hydrolyzed by one molecule of the ISWI
complex/min. Bars and vertical lines represent
the average and the standard deviation, respectively, calculated from
three independent experiments. (C–E) The mutant
yeast ISWI complexes are defective in the MNase ladder assay,
restriction enzyme accessibility assay, and nucleosome spacing assay,
respectively. All assays were done in the standard conditions. For
C and E, partial and extended MNase digestions were
performed for each sample.