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Genetic Dissection of the Function of Histone Variant H2A.Z in Saccharomyces Cerevisiae

Jensen, Kurt
Thesis/Dissertation; Online
Jensen, Kurt
Kupfer, Gary
Brown, Jay
Grant, Patrick
Beyer, Ann
Engel, Daniel
Smith, Mitch
Nucleosomes create a barrier to DNA - dependent processes, and the eukaryotic cell uses several mechanisms to overcome this repression, including incorporation of histone variants. H2A.Z represents the largest conserved family of variants, and has been linked in many organisms to many processes. Here we present evidence that the conserved acidic patch of Htzl (H2A.Z in Saccharomyces cerevisiae) is crucial for Htzl function. We performed an unbiased mutagenic screen designed to elucidate the function of Htzl in yeast. Two mutants identified in the screen highlighted the conserved acidic patch of Htzl. We show that three single point mutations in the patch are phenotypically null (htzI - D99A, htzI - D99K, htzI - E10IK). These mutant proteins are expressed at lower levels, but are functionally defective. The mutants are significantly less detectable by chromatin immunoprecipitation at PH 05, a gene previously described to be enriched for Htzl. These mutants remain phenotypically null and less detectable in chromatin even when protein abundance has been artificially amplified to wild - type levels. We demonstrate that these mutants purify with the SWR1 complex, the complex normally responsible for incorporating Htzl into chromatin, at a substantially reduced level. We propose that the acidic patch of Htzl is a critical region for mediating SWR1 complex functions, such as binding of Htzl. Note: Abstract extracted from PDF file via OCR
University of Virginia, Department of Microbiology, PHD, 2008
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