Mechanism of Origin Activation by Monomers of R6K-encoded π Protein

One recurring theme in plasmid duplication is the recognition of the origin of replication ( ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, π, with seven tandem iterons of γ ori. Remarkably, both π monomers and π dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which π monomers out-compete π dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that π monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that π bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. π monomer/iteron interactions were quantified using a monomer-biased π variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. π dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that π dimers bind with negligible cooperativity to two tandem iterons.