Dynamic DnaA-DnaB interactions at oriC coordinate the loading and coupled translocation of two DnaB helicases for bidirectional replication.

Bidirectional replication is a conserved principle requiring coordinated translocation of the two replicative helicases loaded at the origin. In Escherichia coli, the initiator protein DnaA constructs two oligomers in an initiation complex at the origin oriC, which locally unwinds the DNA and recruits DnaB helicase-DnaC loader complexes to the unwound region. We previously demonstrated that the two DnaA subcomplexes constructed on oriC bind a specific DNA strand of the unwound origin and tether individual DnaB-DnaC complexes via stable interactions between DnaA domain I and DnaB. A low-affinity DnaA-DnaB interaction mediated by DnaA domain III His136 is essential for DnaB-dependent origin unwinding. Here, we identified DnaB Thr86 as the critical residue mediating this low-affinity interaction. Structural modeling suggests that Thr86 is surface-exposed near the DNA entry site of DnaB. Functional analyses revealed that DnaB Thr86 was specifically required for DnaB loading onto the DnaA-bound strand of the unwound oriC. Furthermore, this strand-specific DnaB loading was required for enabling translocation of the opposing DnaB helicase loaded on the DnaA-free strand. Our findings define a novel mechanism of strand-specific helicase loading, mediated by the low-affinity DnaA-DnaB interactions, which promotes the coordinated translocation of the loaded DnaB helicases, ensuring bidirectional replication from oriC.