ESCHERICHIA-COLI SEQA PROTEIN BINDS ORIC IN 2 DIFFERENT METHYL-MODULATED REACTIONS APPROPRIATE TO ITS ROLES IN DNA-REPLICATION INITIATION AND ORIGIN SEQUESTRATION

The seqA gene negatively modulates replication initiation at the E. co li origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation oc curs exactly once per chromosome per cell cycle. Initiation is promote d by full methylation of GATC sites clustered in oriC; sequestration i s specific to the hemimethylated forms generated by replication, SeqA protein purification and DNA binding are described. SeqA interacts wit h fully methylated oriC strongly and specifically, This reaction requi res multiple molecules of SeqA and determinants throughout oriC, inclu ding segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequ ences are bound similarly. Also, binding of hemimethylated oriC by mem brane fractions is due to SeqA. Direct interaction of SeqA protein wit h the replication origin is likely to be involved in both replication initiation and sequestration.