Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC

Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.