Investigating the DnaA-trio, a new essential bacterial replication origin element that specifies single-stranded DNA initiator binding

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In all cells genome duplication requires key proteins ("initiators") to unwind the DNA double helix at specific sites ("origins"). DNA duplex unwinding provides the replication machinery access to single strands, which act as templates for new rounds of DNA synthesis. While initiators are highly conserved throughout all organisms and well characterised, origins are enigmatic. In fact within higher organisms, such as humans, we have yet to define what constitutes an origin. 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. Recently we reported the identification of a novel replication origin element (termed the "DnaA-trio") and showed that it acts to promote single-strand DNA binding activity of the master bacterial replication initiator protein, DnaA. We proposed that this essential recognition element represents a conserved component of the core bacterial chromosome origin. One of the goals of this proposal is to determine how widespread the DnaA-trio is within the bacterial kingdom. We are particularly interested in testing whether this chromosome origin element is present and active in human pathogens such as Staphylococcus aureus and Helicobacter pylori, since this would identify a novel target for antibiotic development. Another goal is to characterize the interaction of DnaA-trios with the initiator protein DnaA. Further understanding of the molecular mechanisms underlying DnaA recognition and activity with this new chromosome origin element will provide cutting-edge knowledge regarding a fundamental biological process that is essential for viability and proliferation. It is important to stress that all initiator proteins throughout the three kingdoms of life contain a related protein fold (the initiator specific AAA+ motif), indicating that they evolved from a common ancestor and that they share common activities. Therefore, the findings from this research project will inform how eukaryotic initiator proteins act and will underpin the search for replication origin elements in higher organisms, which at this moment are ill defined.