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

TY - JOUR

T1 - Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

AU - Syeda, Aisha Haneea

AU - Wollman, Adam

AU - Hargreaves, Alexander Leighton

AU - Howard, Jamieson Anthony Leyland

AU - Bruning, Jan-Gert

AU - McGlynn, Peter

AU - Leake, Mark Christian

N1 - © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

PY - 2019/7/9

Y1 - 2019/7/9

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85068169638&partnerID=8YFLogxK

U2 - 10.1093/nar/gkz298

DO - 10.1093/nar/gkz298

M3 - Article

C2 - 31028385

SN - 0305-1048

VL - 47

SP - 6287

EP - 6298

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 12

ER -