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

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.

The start of DNA replication in bacteria requires the multidomain initiator protein DnaA. DnaA binds to specific sequences (DnaA-boxes) within the bacterial origin (oriC) and forms a nucleoprotein complex that acts to separate the two strands of the DNA duplex. Structural studies indicate that DnaA assembles into an ATP-dependent filament, built upon inter-subunit contacts between adjacent AAA+ motifs. It was generally envisioned that DnaA monomers first bound to individual DnaA-boxes within the replication origin, and that these served as a platform for the recruitment of additional proteins for filament construction. However, bacterial replication origins are highly diverse with a variable number and composition of DnaA-boxes. Therefore, it was unknown which DnaA-boxes were critical to promote filament formation and how DnaA filaments were localized at the replication origin.

Recently my laboratory discovered a new essential bacterial replication origin element composed of a repeating trinucleotide motif that we termed the DnaA-trio. We showed that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thereby providing specificity to the ssDNA binding mechanism. We also found that specific DnaA-boxes proximal to the DnaA-trios are required for loading the DnaA filament. Bioinformatic analysis detects this arrangement of binding elements in chromosome origins throughout the bacterial kingdom, suggesting that they are conserved components of the core replication origin.

The specific aims of this project are to determine whether the DnaA-trio element is conserved in other bacterial replication origins, particularly pathogens, and to characterize the activity of the DnaA-trio. We will determine whether DnaA-trios promote dsDNA destabilization by ssDNA stretching, we will investigate the loading of DnaA from a DnaA-box onto DnaA-trios, and we will probe the specific interaction of DnaA filaments with DnaA-trios.

Antimicrobial resistance is a serious threat to global public health, leading to mounting healthcare costs, treatment failure, and deaths. If unchecked it has been projected that drug resistant infections will kill an extra 10 million people a year worldwide by 2050, more than currently die from cancer, with associated costs spiralling to £63 trillion.

New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. DNA replication is a conserved and essential cellular process and the proteins that replicate DNA in bacteria are distinct from those in eukaryotes; however, none of the antibiotics in current clinical use act directly on the bacterial DNA replication machinery. Bacterial DNA synthesis is therefore an underexploited drug target.

The master DNA replication initiator DnaA is an essential protein present in all human pathogens. Structural studies of DnaA proteins from diverse bacteria have shown that individual domains are homologous, indicating that small molecule inhibitors targeting conserved features would act on a broad range of DnaA proteins.

This research will identify and characterize novel and essential complexes between DnaA and the bacterial chromosome origin that could be inhibited by small molecules. Compounds capable of inhibiting DnaA activity would constitute a new group of antibiotics.