DNA Adenine Methylation Is Required to Replicate Both Vibrio cholerae Chromosomes Once per Cell Cycle
Figure 5
Quantification of hemimethylated DNA in V. cholerae.
(A) Schematic maps of origin regions of the two V. cholerae chromosomes. Both the chromosomal origins (oriI and oriII) are enriched in GATC sites (black dots). Their relative locations are shown with respect to some other features of the origins. For oriI, the features are the flanking genes gidA and mioC, an AT rich region, DnaA boxes (R1–R6) and an IHF site. The features for oriII are described in Figure 3A. Vertical arrows show the two GATC sites studied here for their methylation status. (B) Restriction enzyme names, recognition sequences and their cleavability before and after replication. The recognition sequences are shown in capital letters, and the remainder of the overlapping GATC site is shown in small letters. Prior to replication, these sites are fullymethylated (shown by the attached CH3 group on the adenine residues of both the strands) and are uncleavable (indicated by U); passage of the replication fork generates two hemimethylated products, one remains uncleavable but the other becomes cleavable (indicated by U and C, respectively). Thus, the percent of hemimethylated DNA is twice the percentage of cleavable DNA. (C) Probing of the hemimethylation state of GATC sites located either within the origins (oriI or oriII) or external to the origins (extI or extII) at about 300 kb away. Autoradiographs of Southern blots show sets of three lanes representing repeat experiments from independent cultures. (D) Quantification of band intensities. The bars represent the mean result of the set of three lanes. The experiments were done in LB (black bars) and in MM (M63 medium with casamino acids; gray bars).