In eukaryotic cells, the origin recognition complex (ORC) binds directly to and thereby selects DNA replication origins, the sites on chromosomes where DNA replication initiates. While ORC-origin interactions are fundamental to the duplication and stability of the eukaryotic genome, the molecular mechanisms that regulate these interactions are poorly understood. Our findings in the last grant cycle in Saccharomyces cerevisiae place us in a strong position to address two critical issues relevant to ORC's role in DNA replication initiation. First, we discovered that the established yeast ORC-DNA interface is insufficient to explain ORC binding to a large fraction of origins and that ORC-chromatin interactions contribute more substantially to origin selection in budding yeast than previously appreciated. Thus as proposed for metazoans, an ORC-chromatin interface is important for the selection of DNA replication origins by ORC in budding yeast. We have identified 'chromatin-dependent'yeast origins that will allow us to use the powerful experimental tools of yeast to define the ORC-chromatin interface in molecular detail and thus provide new insights into how ORC selects DNA replication origins within chromosomes. The strong conservation between chromatin, its regulators and the DNA replication proteins across eukaryotes means that substantial aspects of the ORC-chromatin interface we define in budding yeast will be relevant to the analogous interface in human cells and/or useful for manipulating this interface for improving human health. Second, increasing lines of evidence from a variety of systems indicate that ORC has a more dynamic relationship with chromosomes and origins than commonly depicted or appreciated. Moreover, data suggest that these dynamics are relevant to origin regulation in vivo. These data and our own recent observations focused on the function of 'chromatin-dependent'origins lead us to the following hypothesis: Differences in ORC-origin dynamics, which are caused by differences in ORC-chromatin and ORC-DNA interactions at individual origins, contribute to regulating differences in origin activation that exist among the 100's to 1000's of origins that are used to replicate a eukaryotic genome. We will test this hypothesis and thereby gain new and important insights into the basic but relatively unexplored issue of how ORC-origin interactions regulate origin activation in vivo.
A defining requirement for life is the timely and accurate reproduction of the cellular genome-even minor perturbations of single steps in this complex, multi-step process can destabilize a cell's genome leading to birth defects, inherited diseases or cancer. Genome duplication requires hundreds of proteins that must work together precisely with each other and with the genome itself. This proposal aims to understand how the conserved proteins that control the key first step of genome duplication in eukaryotic cells function and are regulated to promote and maintain healthy cellular life and identity.
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