The long term goal of this research is to understand how domains of eukaryotic chromosomes are assembled into specific chromatin structures that regulate transcription. The proposal is focused on the HMR locus of S. cerevisiae, a locus that is kept transcriptionally repressed by a mechanism known as silencing. Silencing requires the establishment and subsequent maintenance of restricted domains of repressive chromatin structure. At the HMR locus a key step in the establishment of silencing requires that the Sir1 protein associate with a DNA element known as a silencer. The silencer contains binding sites for several ubiquitous proteins, including Rap1p, Abf1p, and ORC, the origin recognition complex. The hypothesis has been presented that Sir1p recognizes and associates with the silencer by binding to silencer-associated proteins such as ORC. Because ORC is presumed to be present at all replication origins, while Sir1 is dedicated to silencing at two loci, HMR and HML, Sir1 is postulated to have very specific interactions with ORC. The experiments proposed in this application are designed to define the mechanisms by which Sir1p recognizes a silencer. A genetic screen has identified sir1srd mutants that are defective in HMR silencer recognition but not in silencing per se. These mutants form the basis of genetic, molecular, and biochemical studies that address several issues relating to Sirp's function in silencing. In the first specific aim, the interaction of Sir1p with ORC will be investigated. Mutations will be made in three ORC genes and tested for their ability to suppress the nonmating defect of sir1srd alleles. Physical interaction between Sir1p and ORC will be examined by co-immunoprecipitation analysis using epitope tagged Sir1p. This analysis will be extended to the Sir1psrd proteins to determine whether the mutant proteins show defective ORC interactions. The possibility that Sir1p interacts with other silencer binding proteins such as Rap1p and Abf1p will also be examined in co-immunoprecipitation assays, and the presence of additional proteins in Sir1p immunoprecipitates will be probed. In the second specific aim, the functional domains of Sir1p that are required for silencer recognition will be identified. The sir1srd screen will be repeated in a sir1 null background to determine whether the N-terminus as well as the C-terminus of Sir1p contains a silencer-recognition domain. The possibility that Sir1p functions as an oligomer at silencers will also be tested using both genetic and immunological approaches. Mutations will be generated in the Sir1p C-terminus, which contains a putative silencer recognition domain, and tested for their ability to complement sir1srd alleles. Interaction of the Sir1 C-terminus with mutant Sir1p proteins will also be tested by co-immunoprecipitation with epitope tagged proteins. In the third specific aim, novel proteins that assist Sir1p to recognize a silencer will be identified using two different genetic screens. Existing dosage suppressors of sir1srd alleles will be characterized more fully for their roles in silencing. Loss-of-function mutations in genes required for Sir1p's recognition of a silencer will be identified in a screen for alpha maters in a specialized genetic background. Genes identified in this screen will be isolated and characterized for their effects on silencing. The functions of ORC in DNA replication and silencing will be examined in the fourth specific aim. The role of flanking HMR-E sequences in ORC function in replication and silencing will be determined through deletion analysis. The efficiency with which HMR-E is used as a replication origin in vivo will be tested by 2D fork migration analysis and related to two models for how ORC might affect both replication and silencing.
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