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Lookup NU author(s): Dr Adam WollmanORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. 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 Escherichia 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.
Author(s): Syeda AH, Wollman AJM, Hargreaves AL, Howard JAL, Bruning J-G, McGlynn P, Leake MC
Publication type: Article
Publication status: Published
Journal: Nucleic Acids Research
Year: 2019
Volume: 47
Issue: 12
Pages: 6287-6298
Print publication date: 09/07/2019
Online publication date: 27/04/2019
Acceptance date: 24/04/2019
Date deposited: 10/02/2020
ISSN (print): 0305-1048
ISSN (electronic): 1362-4962
Publisher: Oxford University Press
URL: https://doi.org/10.1093/nar/gkz298
DOI: 10.1093/nar/gkz298
PubMed id: 31028385
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