SRP176455 PRJNA513325 A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication Through its ability to catalyze de novo and maintenance methylation of adenine bases at palindromic GATC sequences in DNA, the bacterial Dam methylase participates in multiple processes including those of: methyl-directed mismatch repair; initiation of DNA replication both of the circular bacterial chromosome from oriC (which is DnaA-dependent) as well as of different plasmids from their respective ori sites; transient sister chromatid cohesion during replication fork progression; and regulation of gene expression. Here we show that Dam controls aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR) in Escherichia coli. Dam methylase deficiency conferred cSDR and, in presence of additional rpoB*35 and tus mutations (that facilitate of replication fork progression during cSDR), robustly rescued the lethality of ?dnaA mutants. In dam dnaA+ strains, a "mid-terminus peak" in gene copy-number analysis was observed which was similar to that earlier described for rnhA, recG, or topA mutants that also exhibit cSDR. The viability of ?dnaA dam derivatives was dependent on a functional MutHLS system, indicating that double strand ends (DSEs) provoked by mismatch repair in the dam mutant are required for cSDR. Another DSE-generating agent phleomycin was able to rescue ?dnaA lethality at sublethal concentrations, but only so in the dam mutS derivative. The DinG helicase was required for rescue of ?dnaA inviability by dam, rnhA, or recG mutations. Our results suggest that in addition to MutHLS-mediated DSEs, a second mechanism operates in the dam mutant to promote dnaA-independent replication. We propose that at sites of collapsed replication forks and at D-loops, absence of Dam methylation is associated with an increased likelihood of reverse restart, that is, retrograde progression of restarted replication forks on the chromosome.