In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly

Yang Lim; Lukas Tamayo Orrego; Ernst Schmid; Zygimante Tarnauskaite; Olga V. Kochenova; Rhian Gruar; Sachiko Muramatsu; Luke Lynch; Aitana  Verdu Schlie; Paula L Carroll; Gheorghe Chistol; Martin A M  Reijns; Masato T. Kanemaki; Andrew P Jackson; Johannes C. Walter

doi:10.1126/science.adi3448

In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly

Yang Lim, Lukas Tamayo Orrego, Ernst Schmid, Zygimante Tarnauskaite, Olga V. Kochenova, Rhian Gruar, Sachiko Muramatsu, Luke Lynch, Aitana Verdu Schlie, Paula L Carroll, Gheorghe Chistol, Martin A M Reijns, Masato T. Kanemaki, Andrew P Jackson^*, Johannes C. Walter^*

^*Corresponding author for this work

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

Abstract

: CMG (Cdc45-MCM2-7-GINS) helicase assembly is the central event in eukaryotic
replication initiation. In yeast, a multi-subunit “pre-Loading Complex” (pre-LC) accompanies
GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON,
30 a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly
in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by
experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS,
TOPBP1, and DNA pol e. Our evidence suggests that DONSON docks the pre-LC onto MCM2-
7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises
35 CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our
understanding of eukaryotic replication initiation, implicate defective CMG assembly in
microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening
accelerates mechanistic discovery

Original language	English
Journal	Science
Early online date	17 Aug 2023
DOIs	https://doi.org/10.1126/science.adi3448
Publication status	E-pub ahead of print - 17 Aug 2023

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Lim et al-Science-8-8-2023-Manuscript and main figures_reducedsizeAccepted author manuscript, 2.36 MBLicence: Creative Commons: Attribution (CC-BY)

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Lim, Y., Tamayo Orrego, L., Schmid, E., Tarnauskaite, Z., Kochenova, O. V., Gruar, R., Muramatsu, S., Lynch, L., Verdu Schlie, A., Carroll, P. L., Chistol, G., Reijns, M. A. M., Kanemaki, M. T., Jackson, A. P., & Walter, J. C. (2023). In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly. Science. Advance online publication. https://doi.org/10.1126/science.adi3448

@article{66a13e3c8a504db5a4c5eff6de417c68,

title = "In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly",

abstract = ": CMG (Cdc45-MCM2-7-GINS) helicase assembly is the central event in eukaryoticreplication initiation. In yeast, a multi-subunit “pre-Loading Complex” (pre-LC) accompaniesGINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON,30 a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assemblyin vertebrates. Using AlphaFold to screen for protein-protein interactions followed byexperimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS,TOPBP1, and DNA pol e. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises35 CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify ourunderstanding of eukaryotic replication initiation, implicate defective CMG assembly inmicrocephalic dwarfism, and illustrate how in silico protein-protein interaction screeningaccelerates mechanistic discovery",

author = "Yang Lim and {Tamayo Orrego}, Lukas and Ernst Schmid and Zygimante Tarnauskaite and Kochenova, {Olga V.} and Rhian Gruar and Sachiko Muramatsu and Luke Lynch and {Verdu Schlie}, Aitana and Carroll, {Paula L} and Gheorghe Chistol and Reijns, {Martin A M} and Kanemaki, {Masato T.} and Jackson, {Andrew P} and Walter, {Johannes C.}",

year = "2023",

month = aug,

day = "17",

doi = "10.1126/science.adi3448",

language = "English",

journal = "Science",

issn = "0036-8075",

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T1 - In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly

AU - Lim, Yang

AU - Tamayo Orrego, Lukas

AU - Schmid, Ernst

AU - Tarnauskaite, Zygimante

AU - Kochenova, Olga V.

AU - Gruar, Rhian

AU - Muramatsu, Sachiko

AU - Lynch, Luke

AU - Verdu Schlie, Aitana

AU - Carroll, Paula L

AU - Chistol, Gheorghe

AU - Reijns, Martin A M

AU - Kanemaki, Masato T.

AU - Jackson, Andrew P

AU - Walter, Johannes C.

PY - 2023/8/17

Y1 - 2023/8/17

N2 - : CMG (Cdc45-MCM2-7-GINS) helicase assembly is the central event in eukaryoticreplication initiation. In yeast, a multi-subunit “pre-Loading Complex” (pre-LC) accompaniesGINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON,30 a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assemblyin vertebrates. Using AlphaFold to screen for protein-protein interactions followed byexperimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS,TOPBP1, and DNA pol e. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises35 CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify ourunderstanding of eukaryotic replication initiation, implicate defective CMG assembly inmicrocephalic dwarfism, and illustrate how in silico protein-protein interaction screeningaccelerates mechanistic discovery

AB - : CMG (Cdc45-MCM2-7-GINS) helicase assembly is the central event in eukaryoticreplication initiation. In yeast, a multi-subunit “pre-Loading Complex” (pre-LC) accompaniesGINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON,30 a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assemblyin vertebrates. Using AlphaFold to screen for protein-protein interactions followed byexperimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS,TOPBP1, and DNA pol e. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises35 CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify ourunderstanding of eukaryotic replication initiation, implicate defective CMG assembly inmicrocephalic dwarfism, and illustrate how in silico protein-protein interaction screeningaccelerates mechanistic discovery

U2 - 10.1126/science.adi3448

DO - 10.1126/science.adi3448

M3 - Article

SN - 0036-8075

JO - Science

JF - Science

ER -

In silico protein interaction screening uncovers DONSON's role in vertebrate CMG helicase assembly

Abstract

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