PMC

The yeast ISWI complexes have distinct nucleosome spacing activities. (A) Components required for the assembly of regularly spaced nucleosome arrays using the yeast ISWI complexes. Presence (+) and absence (−), respectively, of the components are indicated. Partial and extended MNase digestions were taken for each sample. (B) Titration of the yeast ISWI complexes in nucleosome spacing assay. Threefold serial dilution of the yeast ISWI complexes was used. All reactions contained NAP1, core histones, λ DNA, and ATP at standard concentrations. Partial and extended MNase digestions were performed for each sample.

The yeast ISWI genes show genetic interactions. (A) The synthetic stress-sensitive phenotypes of mutants. Wild-type and mutant cells were cultured to saturation, and tenfold dilutions were made for spotting. The medium and the temperatures used are indicated. Formamide plate, containing 3% formamide in YPD, was incubated at 30°C. Relevant genotypes of the mutants are indicated in on left. (B) Rescue of a synthetic temperature-sensitive phenotype of the isw1 isw2 chd1 mutant. The triple mutant was transformed with the plasmids indicated at left, streaked on synthetic medium lacking uracil, and incubated at the temperatures indicated.

ISW1 and ISW2 complexes have nucleosome-stimulated ATPase activities. ATPase activities of ISW1 and ISW2 complexes were assayed in the presence of buffer (B), free core histones (H), naked DNA (D), or nucleosomes (N). Hydrolysis/complex/min denotes the molecules of ATP hydrolyzed by one molecule of the yeast ISWI complexes/min. Bars and vertical lines represent the average and the standard deviation, respectively, calculated from three independent experiments.

ISW1 complex has nucleosome disruption activity. (A) MNase digestion pattern of chromatin assembled on the plasmid containing the hsp70 gene. Purified NURF, ISW1, and ISW2 complexes were added to plasmid chromatin with or without GAGA factor and ATP. Partial and extended MNase digestions were performed for each sample. Oligonucleotide probes corresponding to the promoter and the distal regions of the hsp70 gene are as described (). (B) Restriction enzyme accessibility assay at the promoter region of hsp70 gene. Percentages of digested templates are indicated at bottom. Oligonucleotide probe used for hybridization is the same as the promoter probe in A.

Yeast has two ISWI genes. (A) Schematic representation of the yeast genes homologous to Drosophila ISWI. Drosophila ISWI is presented at top. Domains with significant homologies are indicated by boxes with the same patterns. The ATPase domains and the carboxy-terminal homologous domains of ISWI proteins are represented by solid and checkered boxes, respectively. The numbers below these domains indicate the length of the domains and homologies to the corresponding domain of Drosophila ISWI protein. Location of the SANT domain in ISWI proteins and the chromodomain and putative DNA-binding domain in CHD1p are indicated above the proteins. (B) ISW1 and ISW2 proteins form distinct complexes. The ISW1 and ISW2 complexes (Mono Q fractions) were separated through 10% SDS-PAGE and stained by silver. (Arrows) Subunits of the complexes. (●) Minor bands that are likely due to proteolytic degradation because the intensity of the bands increases during freeze and thaw. (Asterisks) Minor contaminants, which are present in some, but not in other, preparations (see Fig. A).

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.