The long-term objective of this project is to understand the impact of chromatin dynamics on cell cycle progression. Chromatin is remodeled locally during transcription, replication, recombination, and DNA repair and globally during the cell cycle. This project will focus on the yeast ATP-dependent chromatin remodeler, RSC, in regulating the chromosomal structural events that occur during chromosome segregation, including the cohesion and condensation of sister chromatids and the maintenance of genome integrity. The metazoan counterparts, PBAF and hSWI/SNF, function in related cellular pathways and perturbations are tightly linked to human disease and cancers, underscoring the critical importance of remodeling factors in normal cellular physiology. In the first specific aim, roles for RSC in the cohesion and condensation of sister chromatids for chromosome segregation will be assessed using a combination of in vivo chromatin immunoprecipitation (CHIP) assays, live-cell analysis of chromosome movement, chromosome spread, and genetic assays. The recent discovery that RSC cycles on and off chromatin in a cell cycle-dependent manner will be exploited to identify polypeptides that physically associate with the chromatin-bound RSC complex, directly linking RSC to cellular functions. In the second specific aim, cell cycle-dependent targets of RSC will be identified in a genome-wide localization analysis and the chromatin environment of these sites examined to test the hypothesis that RSC binding is correlated to specific structure. In addition, the rsc mutant hypersensitivities to several genotoxic agents, suggesting a direct or indirect role for RSC in maintaining genome integrity, will be pursued by assaying for completion of DNA repair pathway functions in rsc mutants, and for interactions with mutants defective in distinct repair mechanisms. The hypothesis that RSC is specifically recruited to sites of DNA breaks will also be tested. The experiments proposed in the third aim will examine the intracellular signaling pathways that regulate RSC in genomic transmission. Sfhlp phosphorylation sites determined by mass spectrometry will be mutated and the cellular consequences for chromosome segregation tested. The G1-specific kinase that phosphorylates Sfh1 p will be sought in a kinase chip assay. A coupled genetic screen will identify proteins that mechanistically link the PKC1 pathway and RSC in genomic transmission.
