Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.