Project Summary: A long-term goal of my research program is to understand the molecular mechanisms underlying the initiation of cellular DNA replication. Initiation is a defining commitment to cell proliferation; inappropriate onset of replication can lead to changes in gene copy number, DNA damage, and genetic instabilities. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, our understanding of initiation processes is insufficiently complete to advance such efforts. The present application focuses on the initiation of DNA replication in bacteria. Although a basic framework for this process has been in place for more than 25 years, its mechanistic principles have remained highly enigmatic. By employing an innovative mix of structural methods, new biochemical assays, and analytic technologies, we will answer fundamental questions involving how initiation proteins collaborate to open a bubble in a replication origin and deposit ring-shaped helicases onto the DNA. We will determine in molecular detail: 1) how the DnaA initiator melts DNA and whether nascent single DNA strands subsequently regulate DnaA activity, 2) whether the hexameric DnaB helicase autoregulates its DNA unwinding activity, and 3) how the DnaC helicase loader, together with DnaA and the DnaG primase, systematically cooperate in shepherding the helicase onto DNA. The outcome of the proposed studies will be a stepwise structural and functional picture of the major steps involved in converting a duplex chromosomal region into a bidirectional replication fork. These findings in turn will: 1) define new principles for both the field of DNA replication and the broadr action of ATP-dependent machines and switches, and 2) establish new assays and models for advancing drug-discovery efforts that target initiation systems.