Mechanism of recruitment of DnaB helicase to the replication origin of theplasmid pSC101

Although many bacterial chromosomes rei quire only one replication initiato r protein, e.g,, DnaA, most plasmid replicons depend on dual initiators: ho st-encoded DnaA and plasmid-encoded Rep initiator protein for replication i nitiation. Using the plasmid pSC101 as a model system, this work investigat es the biological rationale for the requirement for dual initiators and sho ws that the plasmid-encoded RepA specifically interacts with the replicativ e helicase DnaB, Mutations in DnaB or RepA that disrupt RepA-DnaB interacti on cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo, Although, interaction of DnaA with DnaB could not subs titute for RepA-DnaB interaction for helicase loading, DnaA along with inte gration host factor, DnaC, and RepA was essential for helicase loading. The refore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helica se appears to have at least a. limited number of nonoverlapping surfaces, e ach of which interacts specifically with a different initiator protein.