Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2.
The yeast SWI2/SNF2 polypeptide is a subunit of the SWI/SNF protein complex that is required for many transcriptional activators to function in a chromatin context. SWI2 is believed to be the founding member of a new subfamily of DNA-stimulated ATPases/DNA helicases that includes proteins that function in DNA repair (RAD5, RAD16, ERCC6), recombination (RAD54), transcription (MOT1, ISWI, brm, BRG1, hBRM) and cell cycle control (STH1). We have created a set of 16 mutations within the SWI2 ATPase domain and have analyzed the functional consequences of these mutations in vivo. We have identified residues within each of the seven ATPase motifs that are required for SWI2 function. We have also identified crucial residues that are interspersed between the known ATPase motifs. In contrast, we identify other highly conserved residues that appear to be dispensable for SWI2 function. We also find that single amino acid changes in ATPase motifs IV and VI lead to a dominant negative phenotype. None of the 12 SWI2 mutations that disrupt SWI2 activity in vivo alter the assembly of the SWI/SNF complex. These studies provide an invaluable framework for biochemical analysis of the SWI2 ATPase and for functional analysis of other SWI2 family members.
Richmond
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Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01605, USA.
Peterson
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010940:Plant Proteins / Q000235:genetics; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins; C499336:SMARCA1 protein, human; C084011:SMARCA2 protein, human; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C078729:sucrose transport protein, plant; D004247:DNA; D000251:Adenosine Triphosphatases; C084108:SMARCA4 protein, human; C499333:SNF2 protein, S cerevisiae; D004265:DNA Helicases