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

The Escherichia coli SeqA protein has been found to affect initiation of re plication negatively, both in vivo and in vitro. The mechanism of inhibitio n is, however, not known. SeqA has been suggested to affect the formation a nd 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 S eqA to oriC by electron microscopy and found that SeqA binds specifically t o 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 i n good agreement with earlier mobility shift studies. The affinity of SeqA for hemimethylated oriC was higher than for fully methylated oriC. The bind ing was in both cases strongly co-operative. 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 additio nal oriC molecules and/or complexes into larger aggregates.