Dimers of π Protein Bind the A+T-Rich Region of the R6K γ Origin near the Leading-Strand Synthesis Start Sites: Regulatory Implications

The replication of γ origin, a minimal replicon derived from plasmid R6K, is controlled by the Rep protein π. At low intracellular concentrations, π activates the γ origin, while it inhibits replication at elevated concentrations. Additionally, π acts as a transcription factor (auto)repressing its own synthesis. These varied regulatory functions depend on π binding to reiterated DNA sequences bearing a TGAGNG motif. However, π also binds to a “non-iteron” site (i.e., not TGAGNG) that resides in the A+T-rich region adjacent to the iterons. This positioning places the non-iteron site near the start sites for leading-strand synthesis that also occur in the A+T-rich region of γ origin. We have hypothesized that origin activation (at low π levels) would require the binding of π monomers to iterons, while the binding of π dimers to the non-iteron site (at high π levels) would be required to inhibit priming. Although monomers as well as dimers can bind to an iteron, we demonstrate that only dimers bind to the non-iteron site. Two additional pieces of data support the hypothesis of negative replication control by π binding to the non-iteron site. First, π binds to the non-iteron site about eight times less well than it binds to a single iteron. Second, hyperactive variants of π protein (called copy-up) either do not bind to the non-iteron site or bind to it less well than wild-type π. We propose a replication control mechanism whereby π would directly inhibit primer formation.