Single-molecule visualization of stalled replication-fork rescue by theEscherichia coliRep helicase

Abstract

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. InEscherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individualE. colireplisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that replication restart is the rate-limiting step of stalled replication rescue.

Escherichia coli DNA replication: the old model organism still holds many surprises

“…In a recent live cell imaging study, it has been proposed that Rep monomers might occupy all six DnaB subunits, and the interaction is stochastic and dynamic (Syeda et al 2019 ). Other studies suggest a lower occupancy (Whinn et al 2023 ). This implies that DnaB may serve as a launching pad for Rep probes, where Rep constantly surveils DNA for protein roadblocks and performs quick displacement (Fig.…”

“…This implies that DnaB may serve as a launching pad for Rep probes, where Rep constantly surveils DNA for protein roadblocks and performs quick displacement (Fig. 1A ) (Syeda et al 2019 , Whinn et al 2023 ). We note in passing that UvrD also colocalizes with the replisome to aid protein displacement, but no specific recruitment factors in this context have been identified (Wollman et al 2024 ).…”

Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines

“…Recently, a large rolling-circle template has been developed and used to visualize collisions between the E. coli replisome and a fluorescently labeled protein roadblock. 199 Traditional long linear DNA templates can be stretched either through manipulation in optical or magnetic tweezers, 200−202 or by the drag force introduced by a laminar flow of buffer. 203,204 To date, most long linear templates use multistep ligations to lambda-DNA.…”

“…Rep transiently interacts with the replisome throughout replication and rapidly removes the dCas9 when the replisome encounters it (Figure 4b). 199 A single dCas9 blocks yeast replisomes with 80−90% efficiency regardless of which DNA strand dCas9 is bound to, showing that the yeast replisome has an intrinsic ability to overcome or remove these roadblocks. 209 Furthermore, the eukaryotic accessory helicase Pif1 can rescue stalled replication forks.…”