DNA replication initiation: mechanisms and regulation in bacteria

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Abstract
Initiator proteins direct the assembly of DNA synthesis enzymes at chromosomal sites in a highly regulated manner. In bacteria, the initiator DnaA cooperatively oligomerizes at the site of replication fork formation to direct melting of DNA duplex strands and loading of the replicative helicase DnaB. DnaA belongs to the AAA+ superfamily of ATPases, and the binding and hydrolysis of nucleotides have a crucial role in controlling DnaA activity. The involvement of DnaA at multiple stages of replication initiation is facilitated by its highly modular domain architecture. Interactions with DnaB are mediated by the extreme N-terminal region, whereas a helix–turn–helix motif located in the C-terminal domain confers the sequence-specific DNA-binding activity needed to recognize the origin of replication. The central region of the protein comprises the highly conserved AAA+ fold and primary oligomerization site. Recent structural studies have helped to reveal how ATP facilitates DnaA auto-assembly. Oligomerized DnaA forms a right-handed helical filament that is stabilized through the formation of a bipartite nucleotide-binding pocket between two successive AAA+ domains. The structural features that allow for DnaA filament formation seem to be conserved among cellular initiators from archaea and eukaryotes. Bacteria use many regulatory strategies that are dedicated to controlling the initiation of DNA replication. Among these, origin sequestration, DnaA titration, dnaA autoregulation and DnaA inactivation closely monitor events that occur at the bacterial replication origin to optimize the intracellular levels and activity of DnaA. The involvement of DNA architectural factors at oriC highlight the cellular context in which initiation takes place. DNA-bending proteins such as Fis (factor for inversion stimulation) and IHF (integration host factor) appear to regulate and fine tune the assembly of DnaA at the replication origin during the cell cycle.