Monomer/Dimer ratios of replication protein modulate the DNA strand-opening in a replication origin

DNA opening is an essential step in the initiation of replication via the Cairns mode of replication. The opening reaction was investigated in a γ ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, π. Reactivity to KMnO4 (indicative of opening) within γ ori DNA occurred in both strands of a superhelical template upon the combined addition of wt π, DnaA and integration host factor (IHF), each protein known to specifically bind γ ori. IHF, examined singly, enhanced reactivity to KMnO4. The IHF-dependent reactive residues, however, are distinct from those dependent on π (wt and hyperactive variants). Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of π were used instead of wt protein. We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by π monomers despite the fact that both monomers and dimers of the protein can bind to iterons (π binding sites). Taken together, our data suggest that π elicits its ability to modulate plasmid copy number at the DNA helix-opening step.