This renewal application proposes to continue studies on the mechanisms for establishment and maintenence of mating type gene silencing in Saccharomyces. Mating type is controlled by the transcriptionally active MAT locus. HML and HMR are cryptic copies of the mating type alleles and contain transcriptionally inactive, or silenced, DNA. Dr. Rine's long range goals are to understand the mechanisms which underly this position effect. How are chromosomes organized into independently regulated domains and what is the relationship between DNA replication and the establishment of these domains? Silencing requires sequence elements, called silencers, two of which flank both HML and HMR. In the case of the HMR-E silencer, several proteins have been shown to bind the silencer and/or be mediators of the silencing process including Rap1p, Rif1p, Abf1p, and the Sir1-4 proteins. DNA replication and silencing are inter-related based on the finding that establishment of silencing requires passage through S- phase. Certain histone H3 and H4 mutations lead to expression of HML and HMR suggesting that silencing involves changes in chromatin structure. Dr. Rine proposes to use both genetical and biochemical approaches to investigate silencing. The relationship between the establishment of silencing and passage through S-phase will be investigated in detail to determine whether DNA replication itself or some other event that occurs during S-phase is required. Additional genes involved in silencing will be identified mainly from information derived from the yeast genome project. The role of ORC, Origin Recognition Complex, in silencing will be explored. Genes encoding the component proteins of ORC will be subjected to mutation analysis. Site-directed mutagenesis will be used to direct alterations to regions of the ORC proteins involved in protein-protein interations. Functional domains involved in silencing and/or replications will be defined. Dr. Rine will determine the means by which Sir1p is recruited to the silencer. Sir1p does not bind DNA but its function is mediated by silencer elements. When tethered to the silencer by means of a DNA- binding domain such as that from Gal4p, certain other requirements of silencing are bypassed. Thus, Sir1p is likely to interact with other proteins. Dr. Rine plans to isolate assembly defective mutation in SIR1 and other genes. An in vitro system will be established to study silenced DNA. Silenced chromatin will be purified. Attempts will be made to reconstitute silenced DNA in vitro using purified proteins and DNA from HMR. Finally, Sir protein homologues from other organisms will be identified and their cytogenetic organization determined.
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