Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis

Using yeast forward and reverse two-hybrid analysis and biochemical techniq ues, we present novel and definitive in vivo and in vitro evidence that bot h the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-enc oded RepA initiator of pSC101. The N-terminal, but not the C-terminal, regi on of RepA interacted with DnaA in vitro. These protein-protein interaction s are critical for two very early steps of replication initiation, namely o rigin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allo wed to associate non-covalently with each other via a pair of leucine zippe rs, replication of the plasmid was supported in vivo. Thus, the result show s that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for rep lication initiation. The results also provide the molecular basis for a nov el, potential, replication-based bacterial two-hybrid system.