The goal of this research is to understand the relationship between chromatin assembly, chromatin structure and nuclear function. It is clear that chromatin assembly is a fundamental process that is essential for the replication and maintenance of the eukaryotic genome. Following DNA synthesis, the genome must be rapidly and accurately assembled into chromatin to re-establish specific chromatin structures onto the daughter DNA strands. Even subtle changes in chromatin structure can lead to gene dysfunction and to disease states, such as cancer. Despite its obvious global importance, the process of chromatin assembly is poorly understood. The research proposed in Specfic Aim 1 will delineate the chromatin assembly mechanism that is mediated by the putative physiologically relevant chromatin assembly factors RCAF (replication-coupling assembly factor) and CAF- 1 (chromatin assembly factor 1). These experiments will utilize a defined recombinant chromatin assembly system that permits us to detect the contribution that RCAF and CAF- 1 make to each step in the process. These biochemical studies of the chromatin assembly mechanism will be complemented by molecular, cellular, and genetic studies to test the hypothesis that RCAF and CAF- 1 are directly involved in the assembly of chromatin in budding yeast (Specific Aim 2). The unique nature of the biochemical system, coupled with the genetic analyses, will provide a coherent and effective approach in this emerging and exciting new area of chromatin assembly, structure and function. In addition, this work will impact the fields of gene expression, DNA repair, replication and recombination and may provide valuable insight into human disease states, including cancer that involve defects in these important chromatin-utilizing processes.
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