The timely and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Accordingly the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long-term objective of this proposal is to determine the events that select the sites of DNA replication and activate the initiation process. The origin recognition complex (ORC) is a key factor controlling these events. ORC is the only S. cerevisiae protein known to specifically recognize all yeast origins of replication. Once bound to these sites, ORC acts to recruit other replication factors to the origin of replication. In this proposal, Dr. Bell will exploit his recent advances in understanding the biochemical functions of ORC to determine how these functions are mechanistically coupled to the replication initiation process. Specifically Dr. Bell will: Determine the mechanism of DNA binding by ORC and how this event alters the function of ORC in the cell. These studies will provide critical information concerning the mechanisms that select origins of replication. Determine the function of ORC during replication complex assembly and activation at origins of replication. Using both biochemical analysis of replication factor interaction and a genetic screen for novel ORC mutants, these studies will define key events required for the assembly of essential replication complexes. Determine the influence of nucleosomes on ORC and origin function. Using nucleosome assembly reactions and a novel genome-wide assay for determining the sites of ORC binding, Dr. Bell will dissect the impact of nucleosomes and chromatin on DNA replication. The highly conserved nature of eukaryotic DNA replication strongly argues that any new understanding of the fundamental mechanisms of eukaryotic DNA replication in yeast will be readily translated to our understanding of the same process in human cells. The rapid progress that can be made in S. cerevisiae make it an ideal organism to perform these studies. New understanding of these proteins will lead to strong candidate targets for anti-fungal compounds and potential markers to identify cancer cells. In addition, the understanding of the yeast replication proteins gained in these studies will direct studies to identify inhibitors of the human analogs of these proteins, which represent important targets for novel chemotherapeutic compounds.
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