Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli.

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation in Escherichia coli has been that to obtain mutants which survive loss of DnaA. Here, we show that a ΔrecD mutation, associated with attenuation of RecBCD's DNA double strand end-resection activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5'-3' single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. One-ended DSBs in the ΔrecD mutant did not rescue ΔdnaA lethality. With two-ended DSBs in the ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that nuclease-mediated DNA resection activity of RecBCD is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse, as may be expected to occur during normal replication at the chromosomal terminus region or during repair of two-ended DSBs following 'ends-in' replication.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.