Structural and Functional Analysis of the Initiation of DNA Replication in Bacteria

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The long-term goal of this research is a detailed understanding of the structure and mechanism of action of the macromolecular complexes, which operate at the bacterial origin of DNA replication. Cells initiate replication of chromosomal DNA by assembling large protein complexes at specialized sites termed origins of DNA replication. The complexes at the origin undergo remodeling through the recruitment and dissociation of factors and eventually mature into a pair of fully formed replication forks. In bacteria, the first to protein to bind to the origin is the DnaA protein, five monomers of which interact at distinct sites and form a large nucleo-protein complex. The architecture of this complex is poorly understood. With ATP, this complex promotes the unwinding of regions of the DNA duplex at the origin, which enables loading of the DnaB replicative helicase (assisted by the DnaC protein). Parallels between the assemblies that operate at the origins of DNA replication in bacteria, archaea and eukaryotes are indicated by common mechanistic details and by amino acid sequence similarities in the constituent proteins. These parallels strongly suggest that common structural principles will emerge from studies with the less complicated replication systems offered by bacteria. The PI will perform biochemical, biophysical and structural studies to understand the role of the DnaA protein in the initiation of DNA replication in bacteria. Although the current state of understanding is advanced, several intriguing questions remain unanswered about the architecture and mechanism of action of the DnaA assembly at the origin of DNA replication. This research will be integrated into the scientific and educational activities being performed by the PI within the Department of Molecular and Cellular Biology at Harvard University. These include the education of graduate and undergraduate students and the training of post-doctoral fellows in the fields of experimental biochemistry and structural biology.