Assembly of the bacterial DNA replication initiation complex

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The cell is the basic unit of structure for all living organisms. For a cell to grow and divide it must follow a blueprint that provides the instructions describing how to perform these essential activities. In all cells this information is encoded within DNA. Every time a cell divides it must replicate its DNA and pass on one complete, undamaged copy to each progeny cell. DNA replication has to be tightly controlled to ensure that each newborn cell contains the correct amount of genetic information. If DNA replication is delayed, then upon cell division one daughter cell will fail to inherit a full complement of the genetic information and will be inviable. If DNA replication occurs earlier than needed the cell will contain too many copies of its genes, leading to altered levels of expression which can cause developmental defects.The start of DNA replication requires dedicated replication initiator proteins. Initiator proteins bind to sites termed origins of replication where they act to recruit the DNA replication machinery. Throughout the three kingdoms of life, all initiator proteins contain a related protein fold (the initiator specific AAA+ motif), suggesting that they share common activities required for their activity. Bacteria, with their relatively simple and well characterised structure and physiology, are ideal systems with which to study the molecular mechanisms of DNA replication because they are readily amenable to genetic manipulation and their proteins tend to be tractable subjects for biochemical and structural analyses. The bacterial DNA replication machinery is also an attractive target for potential antibiotics because it is essential for growth and it is currently free from problems of pre-existing resistance.The bacterial DNA replication initiator protein is called DnaA. DnaA binds to specific sequences within the bacterial replication origin and forms a large nucleoprotein complex that separates the two strands of the DNA duplex to initiate the process of genome duplication. Excitingly, a molecular basis for replication origin opening by DnaA is beginning to emerge. Structural studies using x-ray crystallography have found that DnaA assembles into a helical filament that stretches DNA to promote opening of the replication origin. While these structure-based studies are imperative to derive a molecular understanding of DnaA activity, it is important to note that they are limited because they only provide a static image of the dynamic DNA replication initiation reaction. Furthermore, DnaA was not crystallized in the presence of replication origin DNA. Therefore, the structures of DnaA do not reveal how the protein initially assembles into an oligomer at the replication origin, nor do they reveal how DnaA transitions into the conformation that is thought to stretch and open DNA. Previous work in my laboratory has established a novel biochemical assay using purified proteins that detects DnaA helix formation. We have recently improved this methodology and are now able to demonstrate that DnaA adopts at least two distinct helical assembly states specifically at the replication origin.The purpose of this research project is to investigate the pathway of DNA replication initiation by identifying the sequences within the replication origin that are required for assembling the initial DnaA helix and the sequences that are required for promoting the transition between different DnaA conformations. We will utilize genetic approaches to dissect the origin region in vitro and in vivo and then we will utilize our novel helix formation assay to determine how these changes affect DnaA. This project will provide cutting-edge knowledge and will underpin future studies regarding a fundamental biological question that is essential for cellular viability and proliferation.