Asymmetry of Dam remethylation on the leading and lagging arms of plasmid replicative intermediates

In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post-replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half-life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms. We show that remethylation takes place asynchronously on the leading and lagging daughter strands shortly after replication. On the leading arm the fully methylated adenine is restored similar to 2000 bp (corresponding to 2 s) behind the replication fork, while remethylation takes twice as long (at 3500-4000 bp or similar to 3.5-4 s) on the lagging replicative arm. This observation suggests that Dam remethylation of the lagging arm requires ligated Okazaki fragments.