Biochemical and Genetic Analysis of yFACT, A Novel Nucleosome Reorganizing Factor

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DESCRIPTION (provided by applicant): Large eukaryotic genomes must be packaged as chromatin to keep them organized and stable, but this generally inhibits accessibility to the DNA. Controlling transcription and DMA replication therefore requires the ability to selectively overcome this chromatin barrier. We have identified a factor called yFACT in yeast cells that uses a novel mechanism to diminish the inhibitory effects of chromatin. Unlike the better- characterized remodeling factors that use energy derived from ATP hydrolysis to reposition nucleosomes, yFACT changes the structure of individual nucleosomes without hydrolysing ATP or changing the location of the nucleosomes. We call this novel mechanism "nucleosome reorganization" to distinguish it from remodeling. yFACT is required for both transcription and replication. The goal of this proposal is to analyze the mechanism of nucleosome reorganization and to determine how this reorganizing activity participates in a variety of distinct steps in transcription and replication in living yeast cells. yFACT must interact with many different proteins to accomplish these diverse functions. The first aim of the proposal is to better characterize the interaction between yFACT and two of its known partners, RPA and histones, and then to determine how this binding contributes to the range of yFACT functions. RPA is a key factor in DNA replication and histones compose the protein core of nucleosomes, so these interactions are likely to be central to the diverse functions of yFACT. Models for how these interactions contribute to replication, repair, and transcription are proposed and tested. The second aim is to better characterize the mechanism of nucleosome reorganization using purified components. The reorganized state will be probed using nucleases, the stability of reorganized nucleosomes will be tested, and the proximity of components to one another will be mapped. Finally, chromatin immunoprecipitation, tests of genetic stability, and 2-D gels will be used to address how mutations in yFACT components affect key intermediates in transcription and replication in living cells. The ability to make accurate copies of DNA, either as RNA to allow normal cellular metabolism or as DNA for segregation to progeny, is fundamentally important. yFACT is a vital and novel component of both processes, so studying it provides insight into several distinct central pathways simultaneously.