By the same authors

From the same journal

From the same journal

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

Research output: Contribution to journalArticle

Author(s)

Department/unit(s)

Publication details

JournalNucleic Acids Research
DateAccepted/In press - 10 Apr 2019
DatePublished (current) - 24 Apr 2019
Original languageEnglish

Abstract

DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live E. coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. 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 PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one 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 findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

Discover related content

Find related publications, people, projects, datasets and more using interactive charts.

View graph of relations