Characterizing replisome disassembly in human cells

Rebecca M. Jones; Joaquin Herrero Ruiz; Shaun Scaramuzza; Sarmi Nath; Chaoyu Liu; Marta Henklewska; Toyoaki Natsume; Robert G. Bristow; Francisco Romero; Masato T. Kanemaki; Agnieszka Gambus

doi:10.1016/j.isci.2024.110260

Characterizing replisome disassembly in human cells

Rebecca M. Jones, Joaquin Herrero Ruiz, Shaun Scaramuzza, Sarmi Nath, Chaoyu Liu, Marta Henklewska, Toyoaki Natsume, Robert G. Bristow, Francisco Romero, Masato T. Kanemaki, Agnieszka Gambus^*

^*Corresponding author for this work

Cancer and Genomic Sciences

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Abstract

To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.

Original language	English
Article number	110260
Journal	iScience
Volume	27
Issue number	7
Early online date	12 Jun 2024
DOIs	https://doi.org/10.1016/j.isci.2024.110260
Publication status	Published - 19 Jul 2024

Bibliographical note

Acknowledgments
This work was supported by the Lister Award and Wellcome Trust Investigator Award (215510/Z/19/Z) for A.G., BBSRC-funded MIBTP studentship and JSPS Summer program for S.S., and the University of Birmingham. We would like to thank Dr Neville Gilhooly and Dr Marco Saponaro for critical discussions of the manuscript, Dr Aggeliki Skagia for creating the pcDNA5-6HIS-Ubiquitin-K48R plasmid, staff within the microscopy and flow cytometry facilities of MDS (University of Birmingham), and staff within the Core Support team of MDS (University of Birmingham).

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10.1016/j.isci.2024.110260Licence: Creative Commons: Attribution (CC BY)

JonesRM2024CharacterizingFinal published version, 52.3 MBLicence: Creative Commons: Attribution (CC BY)

https://linkinghub.elsevier.com/retrieve/pii/S2589004224014858

Replisome unloading: mechanism and importance for cell biology
Gambus, A. (Principal Investigator)
The Wellcome Trust
1/09/19 → 29/08/25
Project: Research

Cite this

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title = "Characterizing replisome disassembly in human cells",

abstract = "To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.",

author = "Jones, {Rebecca M.} and Ruiz, {Joaquin Herrero} and Shaun Scaramuzza and Sarmi Nath and Chaoyu Liu and Marta Henklewska and Toyoaki Natsume and Bristow, {Robert G.} and Francisco Romero and Kanemaki, {Masato T.} and Agnieszka Gambus",

note = "Acknowledgments This work was supported by the Lister Award and Wellcome Trust Investigator Award (215510/Z/19/Z) for A.G., BBSRC-funded MIBTP studentship and JSPS Summer program for S.S., and the University of Birmingham. We would like to thank Dr Neville Gilhooly and Dr Marco Saponaro for critical discussions of the manuscript, Dr Aggeliki Skagia for creating the pcDNA5-6HIS-Ubiquitin-K48R plasmid, staff within the microscopy and flow cytometry facilities of MDS (University of Birmingham), and staff within the Core Support team of MDS (University of Birmingham).",

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AU - Henklewska, Marta

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AU - Bristow, Robert G.

AU - Romero, Francisco

AU - Kanemaki, Masato T.

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N2 - To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.

AB - To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.

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Characterizing replisome disassembly in human cells

Abstract

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Replisome unloading: mechanism and importance for cell biology

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