Cooperative binding mode of the inhibitors of R6K replication, pi dimers

The replication initiator protein, π, plays an essential role in the initiation of plasmid R6K replication. Both monomers and dimers of π bind to iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication, while π dimers inhibit replication. Recently, it was shown that the monomeric form of π binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers outcompete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role that these interactions may have in the inactivation of γ origin. To examine π dimer/iteron interactions in the absence of competing π monomer/iteron interactions using wild-type π, constructs were made with key base changes to each iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that, in the absence of π monomers, π dimers bind with greater cooperativity to alternate iterons than to adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers.