Chromatin and Cell Cycle Regulation of ORC Function

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DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the molecular mechanisms that regulate the activity of DNA replication origins in S. cerevisiae. The origin recognition complex (ORC) directly binds to and regulates the function of eukaryotic replication origins by participating in the assembly of replication complexes that duplicate the chromosomal DNA. Assembly and activation of origin complexes is regulated by the cell cycle and checkpoints to ensure the accuracy of chromosome duplication prior to mitotic segregation. Chromatin structure also regulates origin function through unknown mechanisms. Our guiding hypothesis is that regulation of the interactions of ORC with replication origins is a critical determinant of subsequent origin activity. Using cellular, biochemical, and genetic analyses, we will characterize the cell cycle interactions of ORC with replication origins in S. cerevisiae, focussing on how the chromatin environment influences ORC-origin interactions and origin function. The specific aims of this research arc to: 1) determine how the characteristics of ORC-origin binding regulate the activity of each origin, 2) elucidate the relationship between DNA replication and chromatin structure by analyzing the structural modifications of histones at differently regulated origins, and 3) characterize these interactions throughout the genome. Strict regulation of the activity of replication origins ensures accuracy in the transmission of the genome to daughter cells, proper regulation of cellular proliferation, and integration of cell duplication with gene expression and cellular differentiation. Understanding how replication origin activity is regulated in all of its contexts has the potential of providing improved methods for the detection and treatment of cancer, as well as elucidating mechanisms of development.