The interplay between replication proteins and silencing proteins in Saccharomyces cerevisiae

The cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae are transcriptionally silenced by the formation of a specialized chromatin structure that requires a cell cycle event between early S phase and G2/M phase to achieve repression. Although replication per se seems not to be essential for silencing, mutations in many proteins involved in DNA replication affect silencing. Four DNA sequence elements (silencers) flank the silenced loci. Each silencer includes a binding site (ACS) for the origin recognition complex (ORC). ORC directly interacts with Sir1, a protein factor involved in silencing of HML and HMR, and ORC is required for Sir1 recruitment to the silencers.;In this study, additional roles for ORC in silencing were discovered. Chromatin Immunoprecipitation (ChIP) analysis revealed that ORC physically interacts with internal regions of HMR. This interaction depended on the presence of silencing proteins and, in part, on the silencer, HMR-I. When HMR was excised from the chromatin using a method for in-vivo recombination, ORC presence at internal regions persisted. Further analysis showed that ORC can be recruited to the silencers in the absence of ACS through its interaction with Sir1.;Also, new mutants of Sir2 were identified that are defective for silencing at the restrictive temperature. The mutations are localized throughout Sir2 and display differential effects on silencing. This set of Sir2 mutants may provide an excellent resource for identifying different functions of Sir2. I also found new replication mutants that affects Sir protein recruitment differently. Examination of these mutants suggested that a higher level of Sir2 recruitment does not necessarily reflect more efficacious silencing.;Finally, working conditions for using nicotinamide to relieve Sir2-dependent silencing were optimized and its effects at different cell cycle stages were studied.