The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Histones are evicted and replaced during all types of polymerase movement, making histone deposition a critical process for all aspects of chromosome biology throughout the cell cycle. In vivo, nucleosome formation requires histone-binding proteins to prevent uncontrolled aggregation of histones and DMA. This proposal is focused on a conserved eukaryotic protein complex important for histone deposition, Chromatin Assembly Factor-1 (CAF-1). CAF-1 interacts with and is stimulated to deposit histones by another histone-binding protein termed Asf1. CAF-1 deposits histones preferentially onto replicating DNA, and thus represent a paradigm for understanding nucleosome formation during S phase of the cell cycle. We will address the following interrelated questions: What auxiliary proteins are required for histone deposition by CAF-1? How is CAF-1 stimulated by Asf1? How is the deposition of different core histone subcomplexes (H3/H4 versus H2A/H2B) coordinated? Are histones exchanged among these complexes, and do multiple assembly complexes contribute histones to the same nucleosome? We will address these questions using biochemical, biophysical, and molecular genetic approaches: We have developed a new in vitro nucleosome assembly assay to facilitate isolation of accessory factors and characterization of reaction intermediates during histone deposition. We will explore how CAF-1 interacts with histones by mapping of interaction sites and analytical ultracentrifugation analyses of CAF-1/histone complexes. We will generate differentially labeled histones to determine whether different complexes contribute to the same nucleosome. Relevance: Inhibition of human CAF-1 results in S phase arrest, apparently due to replication fork collapse. A human Asf1 protein is required for human cellular senescence, a differentiation pathway important for avoidance of tumorigenesis. Therefore, these highly conserved histone deposition proteins directly impact genome stability and growth control, important aspects of human health. The proposed biochemical studies of these proteins are consequently of high priority for cancer research, and these and new proteins discovered in the course of this work may be good candidates in the future for therapeutic intervention.
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