Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates.
To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
M E
ME
Gelbart
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T
Rechsteiner
T J
TJ
Richmond
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T
Tsukiyama
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D002843:Chromatin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D022004:Chromosome Structures / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005821:Genetic Techniques; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
D002843:Chromatin; D004271:DNA, Fungal; D005656:Fungal Proteins; D006657:Histones; C086468:ISWI protein; D009707:Nucleosomes; D011994:Recombinant Proteins; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases