Initiation of DNA Replication at Cell Origins in Yeast

The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome from one cell generation to the next, DNA and its associated chromatin structures must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation in somatic cells or result in mutations in the germ line that can cause many different disorders. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention and diagnosis of proliferation of cancer cells and other proliferative disorders. The initiator protein (ORC) cooperates with a series of DNA replication proteins, including Cdc6, Cdt1 and the MCM2-7 hexamer to establish at origins of DNA replication a pre-Replicative Complex (pre-RC) that facilitates later initiation of DNA synthesis at each origin. Recent progress has determined the mechanism of assembly of the pre-RC and the structure of its components. The proposed research in this application will investigate, using proteins from multiple yeast species and human cells, how origins of DNA replication are specified in these diverse genomes. One of them, the yeast S. cerevisiae has DNA sequence specific origins but the others do not; therefore we propose to determine how these other eukaryotes specify and control DNA replication origins.