Catalytic activity of the yeast SWI/SNF complex on reconstituted nucleosome arrays.
A novel, quantitative nucleosome array assay has been developed that couples the activity of a nucleosome 'remodeling' activity to restriction endonuclease activity. This assay has been used to determine the kinetic parameters of ATP-dependent nucleosome disruption by the yeast SWI/SNF complex. Our results support a catalytic mode of action for SWI/SNF in the absence of nucleosome targeting. In this quantitative assay SWI/SNF and ATP lead to a 100-fold increase in nucleosomal DNA accessibility, and initial rate measurements indicate that the complex can remodel one nucleosome every 4.5 min on an 11mer nucleosome array. In contrast to SWI/SNF action on mononucleosomes, we find that the SWI/SNF remodeling reaction on a nucleosome array is a highly reversible process. This result suggests that recovery from SWI/SNF action involves interactions among nucleosomes. The biophysical properties of model nucleosome arrays, coupled with the ease with which homogeneous arrays can be reconstituted and the DNA accessibility analyzed, makes the described array system generally applicable for functional analysis of other nucleosome remodeling enzymes, including histone acetyltransferases.
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Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA 01605, USA.
Peterson
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D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D009687:Nuclear Proteins / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D000071796:SMARCB1 Protein; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012617:Sea Urchins; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; C066190:SNF5 protein, S cerevisiae; C078562:SWI1 protein, S cerevisiae; C072981:SWI3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D015252:Deoxyribonucleases, Type II Site-Specific; C029792:GTCGAC-specific type II deoxyribonucleases; D000251:Adenosine Triphosphatases; C499333:SNF2 protein, S cerevisiae