RecF protein targeting to post-replication (daughter strand) gaps II: RecF interaction with replisomes

The bacterial RecF, RecO, and RecR proteins are an epistasis group involved in loading RecA protein into post-replication gaps. However, the targeting mechanism that brings these proteins to appropriate gaps is unclear. Here, we propose that targeting may involve a direct interaction between RecF and DnaN. In vivo, RecF is commonly found at the replication fork. Over-expression of RecF, but not RecO or a RecF ATPase mutant, is extremely toxic to cells. We provide evidence that the molecular basis of the toxicity lies in replisome destabilization. RecF over-expression leads to loss of genomic replisomes, increased recombination associated with post-replication gaps, increased plasmid loss, and SOS induction. Using three different methods, we document direct interactions of RecF with the DnaN beta-clamp and DnaG primase that may underlie the replisome effects. In a single-molecule rolling-circle replication system in vitro, physiological levels of RecF protein trigger post-replication gap formation. We suggest that the RecF interactions, particularly with DnaN, reflect a functional link between post-replication gap creation and gap processing by RecA. RecF's varied interactions may begin to explain how the RecFOR system is targeted to rare lesion-containing post-replication gaps, avoiding the potentially deleterious RecA loading onto thousands of other gaps created during replication.

Automated summary

The bacterial RecF, RecO, and RecR proteins form a closely related group involved in loading RecA protein into post-replication gaps. The targeting mechanism that brings these proteins to the appropriate gaps is unclear. However, it is proposed that RecF may interact directly with DnaN and this interaction may be involved in the targeting process. Over-expression of RecF, but not RecO or a RecF ATPase mutant, leads to replicosome destabilization and various negative effects, including loss of replicosomes, increased recombination, plasmid loss, and SOS induction. RecF is found to interact with DnaN beta-clamp and DnaG primase, and these interactions may explain the replisome effects. In vitro studies show that physiological levels of RecF can trigger post-replication gap formation. The interactions of RecF, especially with DnaN, may provide insights into how the RecFOR system is targeted to rare lesion-containing post-replication gaps, avoiding potentially deleterious RecA loading onto other replication gaps.