Competition between the replication initiator DnaA the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E-coli in vitro

Background: Following replication initiation, the replication origin (oriC) in Escherichia roll enters a hemimethylated state at Darn methylation site s which are recognized by the SeqA protein. SeqA binds preferentially to he mimethylated GATC sequences of DNA in vitro, SeqA is essential for the sync hronous initiation of chromosome replication front oriC copies in vivo. Results: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC, (ii) SeqA inhibits th e in vitro replication of a hemimethylated oriC plasmid more efficiently th an the fully methylated, (iii) SeqA inhibits competitive binding of DnaA pr otein to the regions of the hemimethylated oriC plasmid, explaining the mec hanism of its inhibitory effect. The inhibition of DnaA binding by SeqA als o occurs efficiently on a small. hemimethylated oriC fragment containing bo th R1 and M DnaA boxes, but not the 13-mer region. Conclusions: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.