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

Research output: Contribution to journal › Article › peer-review

3 Citations (Scopus)

21 Downloads (Pure)

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 CUL2^LRR1-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
Number of pages	26
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

Data Access Statement

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Aga Gambus ([email protected]). Microscopy data reported in this paper will be shared by the lead contact upon request.

Funding

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). R.M.J. J.H.R. S.S. S.N. C.L. and M.H. generated and analyzed the data; T.N. and M.T.K. assisted with generation of degron cell lines; R.M.J. S.S. and A.G. wrote the manuscript; A.G. R.G.B. and F.R. supervised experimental delivery and provided cell lines; A.G. raised funding and designed and coordinated the project. The authors declare no competing interests.

Funders	Funder number
Japan Society for the Promotion of Science (JSPS)
University of Birmingham
Wellcome Trust	215510/Z/19/Z

Keywords

biochemistry
cell biology
genetics

Access to Document

10.1016/j.isci.2024.110260Licence: CC BY 4.0

RM Jones et al_Characterizing replisome disassembly in human cells
Copyright © 2024 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
Final published version, 5.74 MBLicence: CC BY 4.0

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.",

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AU - Jones, Rebecca M.

AU - Ruiz, Joaquin Herrero

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AU - Nath, Sarmi

AU - Liu, Chaoyu

AU - Henklewska, Marta

AU - Natsume, Toyoaki

AU - Bristow, Robert G.

AU - Romero, Francisco

AU - Kanemaki, Masato T.

AU - Gambus, Agnieszka

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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

Bibliographical note

Data Access Statement

Funding

Keywords

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