Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
ATP dependent chromatin remodelers use the energy from ATP hydrolysis to move, disassemble or alter the composition of nucleosomes. Though all remodelers share a conserved ATP hydrolysis and DNA translocase domain, their biochemical actions and in-vivo characteristics differ because of their subunits and accessory domains in the catalytic subunit that regulate its activity. Understanding how these domains contribute to remodeler regulation in terms of substrate interaction and regulation of the catalytic subunit is therefore important to understanding what causes a remodeler to behave differently, and what are the mechanistic underpinnings of such behavior. In this study we have addressed these questions using the SWI/SNF remodeler from budding yeast (Saccharomyces cerevisiae) to explore how different remodelers compare to SWI/SNF in terms of nucleosome interaction. Using a chemical based histone – remodeler photo-crosslinking and labeling approach, we show that different remodelers contact nucleosomes in patterns unique to their functions, and even remodelers that belong to the same family interact with nucleosomes in a unique manner to accomplish their respective remodeling results. In addition we delineate the functions of the AT hook motifs in the catalytic subunit of SWI/SNF using in-vitro and in-vivo techniques. We demonstrate the necessity of the regulatory action of the motif in the context of SWI/SNF remodeling due to its requirement for efficient ATP hydrolysis by the catalytic domain and therefore efficient remodeling. We also demonstrate for the first time that SWI/SNF in yeast is involved in transcriptional repression with evidence that the AT hook alters SWI/SNF activity at particular genomic regions. Regulation of SWI/SNF activity is an increasingly important topic of study, with mutations that cause SWI/SNF dysfunction being implicated in a large number of cancers and neurological diseases. We attempt to find out the biochemical implications of mutations in the catalytic, SnAC and AT hook motifs with respect to SWI/SNF activity. Taken together, this study provides an insight into some of the different mechanisms in which remodelers are regulated using budding yeast as a model system.
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