Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB-DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB-DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

The DnaA AAA+ Domain His136 Residue Directs DnaB Replicative Helicase to the Unwound Region of the Replication Origin, oriC.

Chromosomal replication initiation requires dynamic mechanisms in higher-order nucleoprotein complexes that are constructed at the origin of replication. In Escherichia coli, DnaA molecules construct functional oligomers at the origin oriC, enabling localized unwinding of oriC and stable binding of DnaB helicases via multiple domain I molecules of oriC-bound DnaA. DnaA-bound DnaB helicases are then loaded onto the unwound region of oriC for construction of a pair of replisomes for bidirectional replication. However, mechanisms of DnaB loading to the unwound oriC remain largely elusive. In this study, we determined that His136 of DnaA domain III has an important role in loading of DnaB helicases onto the unwound oriC. DnaA H136A mutant protein was impaired in replication initiation in vivo, and in DnaB loading to the unwound oriC in vitro, whereas the protein fully sustained activities for oriC unwinding and DnaA domain I-dependent stable binding between DnaA and DnaB. Functional and structural analyses supported the idea that transient weak interactions between DnaB helicase and DnaA His136 within specific protomers of DnaA oligomers direct DnaB to a region in close proximity to single stranded DNA at unwound oriC bound to DnaA domain III of the DnaA oligomer. The aromatic moiety of His136 is basically conserved at corresponding residues of eubacterial DnaA orthologs, implying that the guidance function of DnaB is common to all eubacterial species.