Initiation of DNA replication in Bacillus subtilis: structural studies of the DnaA-DnaD interaction

Replication of genetic information is a vital process across all domains of life. Bacillus subtilis is considered the gram-positive model bacterium for studying DNA replication (Escherichia coli has been studied extensively as the gram-negative model) and is most representative of the ancestral phylum of prokaryotes. DNA replication has three distinct stages; initiation, elongation and termination. Replication initiation is the focus of this research and this process occurs at a single origin conserved throughout bacteria, termed oriC. B. subtilis primosomal machinery is formed of replication initiator proteins DnaA, DnaD and DnaB, the helicase loader DnaI, replicative helicase DnaC and primase DnaG. The role of the initiator proteins is to melt the DNA double helix and enable loading of the hexameric ring helicase onto each strand of DNA for bidirectional replication. Initiation is the first stage in DNA replication and despite its importance the molecular mechanisms of replication initiation remain largely unclear. The work presented in this thesis has focussed on the essential interaction between replication initiator proteins DnaA and DnaD, with an aim to characterise their binding interface and reveal molecular details of their mechanisms of interaction during DNA replication initiation. The direct interaction between isolated DnaA domain I and DnaD DDBH2 domain was detected by NMR spectroscopy which was subsequently used to identify the specific residues involved and characterise the nature of the binding interface. The kinetics of the interaction were investigated by SPR and computational techniques were used to model the DnaA-DnaD complex. This structural characterisation of the DnaA-DnaD interaction provides greater understanding of the molecular mechanisms of DnaA and DnaD during DNA replication initiation.