Syeda, Aisha Haneea orcid.org/0000-0003-3201-4135, Wollman, Adam orcid.org/0000-0002-5501-8131, Hargreaves, Alexander Leighton orcid.org/0000-0001-8802-352X et al. (4 more authors) (2019) Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome. Nucleic Acids Research. pp. 6287-6298. ISSN 0305-1048
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 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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) The University of York > Faculty of Sciences (York) > Physics (York) |
Funding Information: | Funder Grant number BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/P00074611 BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/N006453/1 |
Depositing User: | Pure (York) |
Date Deposited: | 11 Apr 2019 13:30 |
Last Modified: | 22 Jan 2025 00:12 |
Published Version: | https://doi.org/10.1093/nar/gkz298 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gkz298 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:144888 |
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