DNA Replication-Linked Chromatin Assembly in Yeast

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The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Nucleosomes compact the DNA and regulate the accessibility of the genome to all aspects of DNA metabolism. During DNA replication, histones are rapidly assembled onto daughter DNA strands; DNA replication in the absence of histones results in lethality. This ordered deposition is mediated by proteins that bind newly synthesized histones and deposit them onto DNA. Nucleosome assembly proteins include Chromatin Assembly Factor-I (CAF-I) and Asflp, which are ubiquitous among eukaryotic organisms. CAF-I and Asflp act synergistically in vitro to form nucleosomes; in vivo, they build chromatin at specialized regions, such as heterochromatic loci that silence gene transcription. CAF-I is also important for chromatin structure at centromeres. The long-term goals of this work are to understand how CAF-I, Asflp, and other histone deposition proteins function synergically, and how these proteins are differentially regulated at different loci. We study these questions in the budding yeast Saccharomyces cerevisiae, a biochemically and genetically tractable organism. Our specific aims include: 1. Development of reagents to study histone deposition in a defined biochemical reaction. We will test mutant in this system to understand at a mechanistic level silencing phenotypes we have observed. This system will also enable discovery of new proteins that stimulate or regulate histone deposition. 2. Investigation of the special role of histone deposition proteins at centromeres. We will determine which aspects of centromeres. We will determine which aspects of centomeric chromatin are built by histone deposition proteins, and investigate how these are recruited to centromeres. 3. Determination of the cellular distribution of the different histone deposition proteins. We will also explore how proteins involved in DNA damage repair and gene silencing regulate histone deposition differently at different loci. Mechanistic understanding of these highly conserved histone deposition proteins will be applicable to all eukaryotic organisms, including humans. Likewise, discovery of how proteins that sense DNA damage regulate nucleosome formation will improve our understanding of mutagenesis and cancer in metazoans. Proper assembly and function of chromosomes is also important for cell cycle progression and gene expression. Therefore, human homologs of proteins investigated in this proposal are good candidate targets for developing new anti- proliferative drugs.