Chromosome maintenance in Escherichia coli – the role of SeqA in mismatch repair

To achieve faithful transfer of the genetic information from one generation to the next, bacteria have evolved different chromosome maintenance systems. In Escherichia coli two such chromosome maintenance systems are based on the DNA sequence motif GATC, which is methylated at the N6 position of the adenines by the DNA adenine methyltransferase (Dam). During DNA replication, unmodified nucleotides are incorporated into the newly synthesized DNA strand, which results in a hemi-methylation of the GATC motifs for a short period of time after the passing of the replication fork. These hemi-methylated GATC motifs are targeted by MutH, a component of the mismatch repair system in E. coli, in order to enable efficient mismatch repair. Hemi-methylated GATCs are further targeted by SeqA, which sequesters the replication origin and is thought to act in chromosome organization and segregation. These chromosome maintenance systems have been studied individually for many years, but little is known about their interplay. As SeqA and MutH target the same DNA motif, the question arises how these factors functionally interact with one another.

We designed several experimental approaches to analyze the functional interaction between MutH and SeqA in E. coli, by investigating the efficiency of mismatch repair in presence and absence of SeqA. In addition to deletion mutants we designed and constructed synthetic secondary chromosomes. Based on the arrangement of GATC sites on these replicons, the binding of only MutH or both MutH and SeqA or none of these proteins is expected. The respective mutation rate should allow to assess the role of SeqA in mismatch repair. First mutation accumulation assays and next generation sequencing experiments showed that the mutation rates in a seqA deletion mutant are slightly increased compared to the wildtype. To support these results, further mutation accumulation assays are performed analyzing the synthetic secondary chromosomes.