Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.