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 enabled the assembly of the pre-RC in vitro with purified proteins. The proposed research in this application will investigate, using the yeast S. cerevisiae and human cell proteins, how the initiation of DNA replication occurs following pre-RC assembly and how this process is regulated by the Cdc7-Dbf4 (DDK) protein kinase and by an intrinsic inhibitor of initiation of DNA replication within the Mcm4 subunit of the MCM2-7 complex. Biochemical and structural studies will elucidate how molecular machines that are driven by ATP function to load ring shaped protein onto double stranded DNA and how origin recognition has evolved in eukaryotes.