Mechanistic Studies of Replication Initiation in Prokaryotes

A long-term goal of our research is to define the molecular mechanisms underpinning the initiation of cellular DNA replication. Initiation represents a central commitment to cell proliferation; inappropriate onset of replication can lead to genetic instabilities, DNA damage, and changes in gene copy number. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, a molecular-level understanding of initiation factors and their activities is insufficiently complete to advance such efforts. The present renewal application focuses on the initiation of DNA replication in bacteria. Although a basic biochemical framework for this process is in place, the mechanistic and regulatory principles by which replication complexes are assembled remain highly enigmatic. In the past project period, we answered long-standing questions about how the bacterial replicative helicase is physically loaded onto DNA and how ATP turnover allosterically controls this process. We uncovered a new conformational switching mechanism in the replicative helicase that controls the binding of primase, which synthesizes short RNAs to jump-start DNA synthesis. We developed new insights into how hexameric helicases use ATP to drive nucleic acid translocation and how small molecules and partner proteins can control these enzymes. Our past progress paves the way for us to tackle exciting new problems involving initiation. Using structural, biochemical, and single-molecule approaches, we will address fundamental questions regarding the structural organization of early-stage replisome formation, the molecular means by which initiation factors exchange partner proteins in an appropriate temporal order, and the ability of replicative helicases to navigate duplex nucleic-acid roadblocks. The outcome of the proposed studies will be a mechanistic picture of the major steps involved in converting a duplex chromosomal region into an elongating replication fork. These findings will define new principles for the field of DNA replication and the broader action of ATP-dependent machines and switches; it will also establish new insights and approaches for advancing drug-discovery efforts that target bacterial initiation systems.