Protein-DNA interactions involved in the initiation of DNA replication
DNA synthesis is a complex process that is regulated at the stage of initiation of DNA replication. In bacteria, replication initiation occurs at the origin of replication (ori), where multiple molecular interactions occur. Our model system to study replication initiation is the Escherichia coli plasmid, R6K. The initiation of R6K replication occurs at γ ori, where there is an A+T rich region followed by seven binding sites for the replication initiator protein, π. π has two functions: monomers of the protein activate γ ori while dimers inhibit replication. We hypothesize that variations in the DNA sequences of the seven π -binding sites (iterons) at γ ori and an eighth iteron downstream of γ ori are important for π monomer and/or dimer binding. To test this hypothesis, the affinity of each of the different iterons for π was studied both in vivo and in vitro. The in vivo assay monitored the relative affinity of each iteron for π in competition with the full γ ori. In the in vitro assay, electrophoretic mobility shift assay was used to quantify π binding affinity with or without a competitor iteron. Results suggested that π binds to different iterons with different affinities, with strongest binding to the iteron having the most consensus sequence, and weakest binding to the iteron with the least consensus sequence.