Abstract
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.
Highlights
Replication arrest is a recognized source of genetic instability in all organisms
Replication and transcription take place simultaneously, and in eukaryotes house-keeping genes are expressed during the S-phase; transcription is susceptible to impair replication progression
We show that only one recombination protein is required for growth when the rrn genes are highly expressed: the RecBCD complex, an exonuclease/recombinase that promotes degradation and RecA-dependent homologous recombination of linear DNA
Summary
Replication arrest is a recognized source of genetic instability in all organisms. Proteins that protect, process, and restart arrested replication forks have been identified, and in eukaryotes their action is coordinated with the induction of a check-point response to prevent cell cycle progression until replication resumes [1,2,3,4]. It has been shown that in spite of the existence of several wellcharacterized replication-restart machineries capable of reloading a replisome at a replication fork, which depend on the nature of the obstruction, most often arrested replication forks do not restart [5]. They are first targeted by various enzymes including accessory replicative helicases and recombination proteins [6,7,8]. It appears that different causes of replication arrest trigger different responses, and that arrested replication forks are channeled to various pathways depending on the original cause of arrest
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