Molecular mechanisms for loading the bacterial replicative helicase

Helicase recruitment, loading, and activation demarcates the first stage of assembling the replisome, the machinery that performs DNA replication. Within bacterial cells, helicase loading must be orchestrated at two distinct events: cell cycle mediated DNA replication initiation at the chromosome origin (oriC) and to restart replication at a repaired replication fork. Replication initiation is guided by the master initiator protein DnaA, while replication restart utilises the helicase PriA. Since DnaA, PriA, and the replicative helicase are conserved throughout the bacterial domain, it is likely that the principal events at oriC and a repaired replication fork are shared amongst diverse species. Moreover, because bacterial replication factors are distinct from their analogues in eukaryotes, the bacterial DNA replication machinery is an attractive drug target. Despite years of study, fundamental aspects of DNA replication initiation in bacteria remain poorly understood. Specifically, the architecture of nucleoprotein complexes required to load helicase(s), and the temporal set of activities that replication initiation proteins perform during the helicase loading reaction, are unclear. This project will combine genetics, biochemistry, cryo-electron microscopy, and single-molecule fluorescence microscopy to unravel the molecular mechanisms of bacterial helicase loading, to near-atomic resolution, at both a chromosome origin and a repaired replication fork.