1 Molekylær og generel fysiologi, Department of Science and Environment, Roskilde University2 The Department of Science, Systems and Models, Roskilde University
GATC sequences in the DNA of Escherichia coli and related species are methylated at the adenine residue by DNA adenine methyltransferase (DamMT). These methylated residues and/or the level of DamMT influence initiation of chromosome replication from the replication origin, oriC, which contain an over-representation of GATC sites, in at least two ways. First, full methylation of oriC promotes duplex opening and hence certain oriC mutants are dependent on Dam methylation for initiation. Second, newly replicated and hemimethylated origins, are bound by SeqA (‘sequestered’) and remain inactive for about one third of the cell cycle. During sequestration at least three mechanisms operate to lower the activity of the initiator protein, DnaA. First, the dnaA promoter, which also contains an excess of GATC sequences, is sequestered for the same period of time as oriC to prevent de novo DnaA synthesis. Second, new DnaA binding sites outside oriC are generated by replication which serve to titrate free DNA protein. Third, after initiation, DnaA-ATP is converted to inactive DnaA-ADP by a process called RIDA (regulatory inactivation of DnaA), which is dependent on the beta-clamp of DNA polymerase III holoenzyme and the Hda protein. Overall these processes contribute to limit chromosome replication to once and only once per cell cycle. Cells deficient in RIDA (by deletion of the hda gene) overinitiate chromosome replication, grows poorly and rapidly accumulate secondary mutations. We analyzed a number of these by whole genome sequencing. In one case the suppression was due to deletion of two Ts upstream of the ybfF gene. This led to increased SeqA production and presumably prolonged hemimethylation. The increased SeqA level affected replication initiation in two ways not described previously. First, rejuvenation of DnaA-ADP to DnaA-ATP by the DARS1 sequence was inhibited. Second, excess SeqA reduced the overall level of DnaA by about 30% through dnaA gene transcription. However, this reduced initiator level did not negatively affect initiation of replication. This suggests that the amount of DnaA available for initiation is not affected by additional SeqA whereas DnaA binding to sites outside the origin is inhibited by increased sequestration and/or hemimethylation.
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6th New England Biolabs Meeting on DNA restriction and modification, 2010