Assembly of the bacterial DNA replication initiation complex

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.

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) where it acts to separate the two strands of the DNA duplex. Structural studies indicate that DnaA assembles into an ATP-dependent helical filament, built upon inter-subunit contacts between adjacent AAA+ motifs, which binds and stretches single-stranded DNA in a manner that prevents pairing with the complimentary strand. However, it remains unclear how DnaA initially assembles into an oligomer and how it transitions into the conformation that interacts with a single-strand of the DNA duplex specifically at oriC.

The purpose of this research proposal is to investigate the pathway of DnaA assembly at oriC using a novel cross-linking assay developed in my laboratory that detects helix formation of the initiator protein. Our preliminary data suggests that DnaA initially binds to duplex DNA within oriC through its C-terminal domain (domain IV) and assembles into a helical oligomer where domain IV would need to be extended away from the AAA+ core of the protein complex. Subsequently, this extended DnaA oligomer supports the assembly of a second distinct helical filament that is capable of binding to one strand of the DNA duplex through residues in the adjacent AAA+-containing domain (domain III).

The specific aims of this research proposal are: to use a reverse genetic approach to dissect the oriC region and elucidate the sequence motifs and properties that are specifically required for DnaA helix formation; to determine the location within oriC where distinct DnaA helical oligomers are assembled; and to determine the biological relevance of oriC sequences required for DnaA helix formation.

The training received by the Research Assistant assigned to this project will be readily transferable to related fields across the spectrum of Molecular Biology, thus facilitating their potential employment either in academia or industry.

Development of information arising from this project will have both short- and long-term impacts for the United Kingdom. In the near-term this new technology could be commercialized and subsequently utilized to identify small molecule inhibitors targeting the essential bacterial DNA replication machinery, thereby addressing the growing problem of antimicrobial resistance to currently available antibiotics and drugs. In the long-term any advances regarding identification of novel antibiotics will benefit the population by helping to combat infectious disease. Decreasing illness will help maintain high levels of worker productivity and reduce the burden of costs to the National Health Services (~10% of its annual budget).