Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
Chromatin remodelers are ATP-dependent multisubunit assemblies that regulate transcription and other processes by altering DNA-histone contacts. The mechanism of action is based on the transduction of energy released by ATP hydrolysis to translocation on DNA and ultimately the movement of histones in cis or trans. Though the critical ATP burning and translocation activities are fulfilled by a conserved ATPase domain in the catalytic subunit, there are accessory domains and subunits that are speculated to regulate these activities. Important questions in the field center around the identification of these domains and subunits, whether they affect complex formation, substrate affinity or a critical step in remodeling. If they do affect remodeling, what is the structural basis of the regulatory activity. In this study, these questions have been addressed using the prototype remodeler SWI/SNF from budding yeast. ySWI/SNF is a 12 subunit complex that includes the catalytic subunit Swi2/Snf2. It affects 6% of the yeast genome being primarily involved in gene activation. We employed a systematic protein or domain deletion strategy and characterized the mutant complexes in vitro and in vivo. A key finding was that SWI/SNF is organized in distinct structural modules and that the Snf2 module regulates most of its activities. Snf2 is a central subunit in this module and the function of conserved regions within Snf2 were studied. The N terminus preceding the HSA and ATPase domain has three major roles - complex assembly, recruitment and regulation of catalytic activity. A novel SnAC domain located C terminal to ATPase domain was identified to play critical role in coupling ATP hydrolysis to nucleosome movement by acting as a histone anchor. Finally the tandem AT-hooks between SnAC and bromodomain serve as DNA binding domains but also affect ATPase activity and nucleosome mobilization independent of its binding activity. Taken together, this study provides a comprehensive overview of the function of regulatory domains in SWI/SNF.
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