Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep–DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle