Protein–DNA complexes are the primary sources of replication fork pausing in Escherichia coli

Abstract

Replication fork pausing drives genome instability, because any loss of paused replisome activity creates a requirement for reloading of the replication machinery, a potentially mutagenic process. Despite this importance, the relative contributions to fork pausing of different replicative barriers remain unknown. We show here that Deinococcus radiodurans RecD2 helicase inactivates Escherichia coli replisomes that are paused but still functional in vitro, preventing continued fork movement upon barrier removal or bypass, but does not inactivate elongating forks. Using RecD2 to probe replisome pausing in vivo, we demonstrate that most pausing events do not lead to replisome inactivation, that transcription complexes are the primary sources of this pausing, and that an accessory replicative helicase is critical for minimizing the frequency and/or duration of replisome pauses. These findings reveal the hidden potential for replisome inactivation, and hence genome instability, inside cells. They also demonstrate that efficient chromosome duplication requires mechanisms that aid resumption of replication by paused replisomes, especially those halted by protein-DNA barriers such as transcription complexes.DNA repair | genome stability | Rep | RNA polymerase | recombination

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

“…Recent biological data have suggested that protein-DNA complexes are the primary source of replication fork pausing in E. coli (46). Emerging evidence supports a model in which replication collisions are overcome by accessory helicases that assist the replisome to progress through protein-DNA blockades (32).…”

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

“…It was recently shown that D. radiodurans RecD2 causes collapsed E. coli replication forks to fail to reactivate in vitro and in vivo (64). Interestingly, D. radiodurans RecD2 overexpressed in E. coli was lethal to ⌬rep helicase mutants.…”

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…Consistent with RF pausing at protein-DNA complexes being a universal threat to genome stability, Escherichia coli also possesses accessory DNA helicases that act to clear protein-DNA barriers ahead of RFs 14 . Indeed, protein-DNA complexes are frequent sources of RF pausing in this organism 15 . Interestingly, E. coli also exhibits at least one example of programmed RF pausing, and this process is required to coordinate efficient DNA replication termination and chromosome segregation.…”