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

Abstract

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 β-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.

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

“…The involvement of homologous recombination to process the unwanted DSB is the most obvious hypothesis and would allow a mismatch repair without strand discrimination. The recruitment of homologous recombination to DNA damage could be achieved via the replication clamp which is a known platform that recruits numerous DNA-processing enzymes including DNA repair enzymes ( 92 ). Besides, unlike most bacteria, Streptomyces species also have the ability to repair DSBs by illegitimate recombination pathway.…”

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

“…Interestingly, in human cells, it has been proposed by Chen et al (87) that PCNA loading on DSBs appearing independently of replication, and functions as a processivity factor for the Exo1 nuclease in DNA resection, thus directly linking DSB repair and homologous recombination mechanism to the sliding clamp. More recently, it was shown in E. coli that RecF targeting to post-replicative gaps involves a direct interaction with DnaN/β-clamp (88). Single-strand DNA post-replication gaps are frequently formed during replication and are predominantly repaired by homologous recombination.…”

Insight into Single-Molecule Imaging Techniques for the Study of Prokaryotic Genome Maintenance

“…75 In a more recent study by Henry et al in 2023, it was proposed that RecF's interaction with the β-clamp potentially triggers the creation of postreplication DNA gaps, which are then processed by the RecFOR system via RecAmediated recombination (Figure 8C). 169 Besides recombination, RecA also orchestrates the cellular response to DNA damage via the bacterial SOS response. 170 The SOS response is a bacterial damage response system triggered by the accumulation of ssDNA when replication stalls at the site of a lesion, while the replicative helicase continues unwinding DNA.…”