The Escherichia coli SeqA protein binds specifically and co-operatively to two sites in hemimethylated and fully methylated oriC.

The Escherichia coli SeqA protein has been found to affect initiation of replication negatively, both in vivo and in vitro. The mechanism of inhibition is, however, not known. SeqA has been suggested to affect the formation and activity of the initiation complex at oriC, either by binding to DNA or by interacting with the DnaA protein. We have investigated the binding of SeqA to oriC by electron microscopy and found that SeqA binds specifically to two sites in oriC, one on each side of the DnaA binding site R1. Specific binding was found for fully and hemimethylated but not unmethylated oriC in good agreement with earlier mobility shift studies. The affinity of SeqA for hemi-methylated oriC was higher than for fully methylated oriC. The binding was in both cases strongly cooperative. We suggest that SeqA binds to two nucleation sites in oriC, and by the aid of protein-protein interaction spreads to adjacent regions in the same oriC as well as recruiting additional oriC molecules and/or complexes into larger aggregates.