Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101.

We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101. We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw. This cooperative interaction at a distance, most probably, caused looping out of the ihf site. We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping. The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site. Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro. On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin.