This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sir3 is involved in the formation of heterochromatin at the silent mating type loci and in subtelomeric regions of S.cerevisiae chromosomes. According to the current model the Sir complex (Sir2, Sir3 and Sir4) is initially brought to a silencer region, through association with silencer binding proteins (probably ORC, Abf1 and Sir1) and then spreads several kilobases from the silencer. Sir bound regions become non-permissive to transcription by RNApolII and histones associated with the Sir complex become deacetylated and demethylated. In order to better understand the mechanisms of Sir complex binding, transcriptional silencing, histone modification changes and Sir complex boundary formation we plan to identify new proteins associated with the Sir3 complex. Sir3 associated proteins will be purified from formaldehyde crosslinked cell lysates with a two step purification procedure using a Sir3 construct with a His tag and a biotinylation signal. Purified peptides will then be subject to mass spec analysis.
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