Effect of altering GATC sequences in the plasmid ColE1 primer promoter

Plasmid ColE1 has three recognition sites for the Escherichia coli DNA adenine methylase in the immediate upstream region of the primer promoter. Two of these sites are conserved among all plasmid relatives of ColE1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. Recent experiments have indicated that hemimethylated ColE1-type plasmids are inefficiently replicated after transformation (D. W. Russell and N. Zinder, Cell 50:1071-1079, 1987). By mutating the three methylation sites, we found that disruption of all three GATC sites was necessary for complete relief of the hemimethylation-mediated inhibition of replication in vivo. We also found that these three methylation sites acted in a position-specific manner. The putative cruciform, if present, did not play a regulatory role in the hemimethylation-mediated inhibition of replication.