The structural mechanism of dimeric DONSON in replicative helicase activation

Miloš Cvetković; Paolo Passaretti; Agata Butryn; Alicja Reynolds-Winczura; Georgia Kingsley; Angeliki Skagia; Cyntia Fernandez Cuesta; Divya Poovathumkadavil; Roger George; Anoop Chauhan; Satpal Jhujh; Grant Stewart; Aga Gambus; Alessandro Costa

doi:10.1016/j.molcel.2023.09.029

The structural mechanism of dimeric DONSON in replicative helicase activation

Miloš Cvetković, Paolo Passaretti, Agata Butryn, Alicja Reynolds-Winczura, Georgia Kingsley, Angeliki Skagia, Cyntia Fernandez Cuesta, Divya Poovathumkadavil, Roger George, Anoop Chauhan, Satpal Jhujh, Grant Stewart, Aga Gambus^*, Alessandro Costa^*

^*Corresponding author for this work

Cancer and Genomic Sciences

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

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Abstract

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients’ mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

Original language	English
Pages (from-to)	4017-4031.e9
Number of pages	24
Journal	Molecular Cell
Volume	83
Issue number	22
Early online date	10 Oct 2023
DOIs	https://doi.org/10.1016/j.molcel.2023.09.029
Publication status	Published - 16 Nov 2023

Bibliographical note

Acknowledgments:
We acknowledge the Crick Structural Biology STP for support with computation (A. Nans, A. Purkiss, and P. Walker) and cryo-EM support (A. Nans and D. Benton). This work was funded jointly by the Wellcome Trust, MRC, and CRUK at the Francis Crick Institute (FC001065). A.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 820102). M.A.C. was supported by a Marie Skłodowska-Curie Postdoctoral Fellowship (agreement no. 845939). A Wellcome Trust Investigator Award (215510/Z/19/Z) to A.G. funded P.P., BBSRC (BB/T001860/1) was awarded to A.G. and funded A.S. and A.R.-W., and MRC funded IMPACT PhD studentship for G.K. and BBSRC MIBTP studentship for C.F.-C. Work by G.S.S.’s group is funded by a CRUK programme grant (C17183/A23303), which also funded S.S.J. An MRC project grant MR/W001152/1 awarded to G.S.S. funded A.S.C.

Keywords

DNA replication initiation
replicative helicase
DONSON
cryo-EM
Xenopus egg extract
primordial dwarfism

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10.1016/j.molcel.2023.09.029Licence: Creative Commons: Attribution (CC BY)

CvetkovicM2023structuralFinal published version, 10.1 MBLicence: Creative Commons: Attribution (CC BY)

1 Finished
3 Active

Investigating the role of arginine methylation as a critical regulator of DNA replication and genome stability
Stewart, G. (Principal Investigator)
Medical Research Council
1/01/22 → 30/06/25
Project: Research Councils
Replisome unloading: mechanism and importance for cell biology
Gambus, A. (Principal Investigator)
The Wellcome Trust
1/09/19 → 29/08/25
Project: Research
Characterising novel regulators of the PI-3-kinase-like kinase-dependent DNA damage response and their role in preventing human disease and cancer
Stewart, G. (Principal Investigator)
Cancer Research Uk
1/09/17 → 31/05/25
Project: Research
TRAIP ubiquitin ligase driving replisome disassembly
Gambus, A. (Principal Investigator)
Biotechnology & Biological Sciences Research Council
1/12/19 → 30/11/23
Project: Research Councils

Cite this

Cvetković, M., Passaretti, P., Butryn, A., Reynolds-Winczura, A., Kingsley, G., Skagia, A., Fernandez Cuesta, C., Poovathumkadavil, D., George, R., Chauhan, A., Jhujh, S., Stewart, G., Gambus, A., & Costa, A. (2023). The structural mechanism of dimeric DONSON in replicative helicase activation. Molecular Cell, 83(22), 4017-4031.e9. https://doi.org/10.1016/j.molcel.2023.09.029

@article{61bd4b775ec14b649c215a9aa1bd88c4,

title = "The structural mechanism of dimeric DONSON in replicative helicase activation",

abstract = "The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients{\textquoteright} mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.",

keywords = "DNA replication initiation, replicative helicase, DONSON, cryo-EM, Xenopus egg extract, primordial dwarfism",

author = "Milo{\v s} Cvetkovi{\'c} and Paolo Passaretti and Agata Butryn and Alicja Reynolds-Winczura and Georgia Kingsley and Angeliki Skagia and {Fernandez Cuesta}, Cyntia and Divya Poovathumkadavil and Roger George and Anoop Chauhan and Satpal Jhujh and Grant Stewart and Aga Gambus and Alessandro Costa",

note = "Acknowledgments: We acknowledge the Crick Structural Biology STP for support with computation (A. Nans, A. Purkiss, and P. Walker) and cryo-EM support (A. Nans and D. Benton). This work was funded jointly by the Wellcome Trust, MRC, and CRUK at the Francis Crick Institute (FC001065). A.C. receives funding from the European Research Council (ERC) under the European Union{\textquoteright}s Horizon 2020 research and innovation programme (grant agreement no. 820102). M.A.C. was supported by a Marie Sk{\l}odowska-Curie Postdoctoral Fellowship (agreement no. 845939). A Wellcome Trust Investigator Award (215510/Z/19/Z) to A.G. funded P.P., BBSRC (BB/T001860/1) was awarded to A.G. and funded A.S. and A.R.-W., and MRC funded IMPACT PhD studentship for G.K. and BBSRC MIBTP studentship for C.F.-C. Work by G.S.S.{\textquoteright}s group is funded by a CRUK programme grant (C17183/A23303), which also funded S.S.J. An MRC project grant MR/W001152/1 awarded to G.S.S. funded A.S.C.",

year = "2023",

month = nov,

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doi = "10.1016/j.molcel.2023.09.029",

language = "English",

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Cvetković, M, Passaretti, P, Butryn, A, Reynolds-Winczura, A, Kingsley, G, Skagia, A, Fernandez Cuesta, C, Poovathumkadavil, D, George, R, Chauhan, A , Jhujh, S , Stewart, G , Gambus, A & Costa, A 2023, 'The structural mechanism of dimeric DONSON in replicative helicase activation', Molecular Cell, vol. 83, no. 22, pp. 4017-4031.e9. https://doi.org/10.1016/j.molcel.2023.09.029

TY - JOUR

T1 - The structural mechanism of dimeric DONSON in replicative helicase activation

AU - Cvetković, Miloš

AU - Passaretti, Paolo

AU - Butryn, Agata

AU - Reynolds-Winczura, Alicja

AU - Kingsley, Georgia

AU - Skagia, Angeliki

AU - Fernandez Cuesta, Cyntia

AU - Poovathumkadavil, Divya

AU - George, Roger

AU - Chauhan, Anoop

AU - Jhujh, Satpal

AU - Stewart, Grant

AU - Gambus, Aga

AU - Costa, Alessandro

N1 - Acknowledgments: We acknowledge the Crick Structural Biology STP for support with computation (A. Nans, A. Purkiss, and P. Walker) and cryo-EM support (A. Nans and D. Benton). This work was funded jointly by the Wellcome Trust, MRC, and CRUK at the Francis Crick Institute (FC001065). A.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 820102). M.A.C. was supported by a Marie Skłodowska-Curie Postdoctoral Fellowship (agreement no. 845939). A Wellcome Trust Investigator Award (215510/Z/19/Z) to A.G. funded P.P., BBSRC (BB/T001860/1) was awarded to A.G. and funded A.S. and A.R.-W., and MRC funded IMPACT PhD studentship for G.K. and BBSRC MIBTP studentship for C.F.-C. Work by G.S.S.’s group is funded by a CRUK programme grant (C17183/A23303), which also funded S.S.J. An MRC project grant MR/W001152/1 awarded to G.S.S. funded A.S.C.

PY - 2023/11/16

Y1 - 2023/11/16

N2 - The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients’ mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

AB - The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients’ mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

KW - DNA replication initiation

KW - replicative helicase

KW - DONSON

KW - cryo-EM

KW - Xenopus egg extract

KW - primordial dwarfism

U2 - 10.1016/j.molcel.2023.09.029

DO - 10.1016/j.molcel.2023.09.029

M3 - Article

C2 - 37820732

SN - 1097-2765

VL - 83

SP - 4017-4031.e9

JO - Molecular Cell

JF - Molecular Cell

IS - 22

ER -

The structural mechanism of dimeric DONSON in replicative helicase activation

Abstract

Bibliographical note

Keywords

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Projects

Investigating the role of arginine methylation as a critical regulator of DNA replication and genome stability

Replisome unloading: mechanism and importance for cell biology

Characterising novel regulators of the PI-3-kinase-like kinase-dependent DNA damage response and their role in preventing human disease and cancer

TRAIP ubiquitin ligase driving replisome disassembly

Cite this