Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

Abstract

AbstractDNA replication requires strategies to cope with nucleoprotein barriers that impair the efficient translocation of the replisome. Biochemical and genetic studies indicate accessory helicases play essential roles in continuity of replication in the presence of nucleoprotein barriers, but how they operate in the native cellular environment is unclear. With high-speed single-molecule microscopy we determine the dynamic patterns of localization of genomically-encoded fluorescent protein constructs of the bacterial accessory helicase Rep and core replisome protein DnaQ in live E. coli cells. We demonstrate that Rep colocalizes with 70% of replication forks. Colocalisation is dependent upon interaction with replicative helicase DnaB, with an underlying hexameric stoichiometry of Rep indicating maximal occupancy of the single DnaB hexamer within the replisome. We find that Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged the PriC replication restart factor given the known Rep-PriC functional interaction and observe Rep-replisome association is also dependent on the presence of PriC. Our findings suggest two Rep-replisome populations in vivo: one involving Rep continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These new findings reveal how a single type of helicase is recruited to the replisome to provide two independent ways of underpinning replication of protein-bound DNA, a problem that all organisms face as they replicate their genomes.Significance statementAll organisms face the challenge of proteins bound to DNA acting as barriers to prevent DNA replication. We have performed fluorescence imaging experiments on living bacteria to track the positions of the replication machinery, a protein called Rep which is involved in removing these barriers, and a protein called PriC believed to be involved with reloading the replication machinery if the original replication machinery breaks down. We find that Rep is very dynamic with continual binding and movement away from the replication machinery. Association with the replication machinery depends on both binding to the replication machinery directly and on PriC. Thus Rep can circumvent barriers in two independent ways: a strategy which may be relevant to all organisms.