A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Humberto Sánchez; Zhaowei Liu; Edo van Veen; Theo van Laar; John F. X. Diffley; Nynke H. Dekker

doi:10.1038/s41467-023-42524-8

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Humberto Sánchez
, Zhaowei Liu
, Edo van Veen
, Theo van Laar
, John F. X. Diffley
, Nynke H. Dekker

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

Original language	English
Article number	6735
Pages (from-to)	1-15
Number of pages	15
Journal	Nature Communications
Volume	14
Early online date	23 Oct 2023
DOIs	https://doi.org/10.1038/s41467-023-42524-8
Publication status	Published - 2023
Externally published	Yes

Funding

We thank Anne Early and Lucy Drury for providing yeast strains, Nerea Murugarren for assistance in loading bulk biochemical assays, Fiona Horne and Emilie van Vet for preliminary chromatin assembly experiments, and Alessandro Costa for useful discussions. Z.L. acknowledges the support given by EMBO Postdoctoral Fellowship (ALTF 484-2022). N.D. acknowledges funding from the Netherlands Organization for Scientific Research (NWO) through Top grant 714.017.002), \u201CBaSyC\u2014Building a Synthetic Cell\u201D Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW), and from the European Research Council through an Advanced Grant (REPLICHROMA; grant number 789267.

Funders	Funder number
European Research Council
Ministerie van Onderwijs, Cultuur en Wetenschap
Horizon 2020 Framework Programme	789267
European Molecular Biology Organization	ALTF 484-2022
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	714.017.002, 024.003.019

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title = "A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint",

abstract = "Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.",

author = "Humberto S{\'a}nchez and Zhaowei Liu and \{van Veen\}, Edo and \{van Laar\}, Theo and Diffley, \{John F. X.\} and Dekker, \{Nynke H.\}",

year = "2023",

doi = "10.1038/s41467-023-42524-8",

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journal = "Nature Communications",

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AU - Sánchez, Humberto

AU - Liu, Zhaowei

AU - van Veen, Edo

AU - van Laar, Theo

AU - Diffley, John F. X.

AU - Dekker, Nynke H.

PY - 2023

Y1 - 2023

N2 - Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

AB - Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

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U2 - 10.1038/s41467-023-42524-8

DO - 10.1038/s41467-023-42524-8

M3 - Article

SN - 2041-1723

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SP - 1

EP - 15

JO - Nature Communications

JF - Nature Communications

M1 - 6735

ER -

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Abstract

Funding

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