Cryo-EM structure of human hexameric MCM2-7 complex

Xu, Naining; Lin, Qingpeng; Tian, Honglei; Liu, Changdong; Wang, Peiyi; Suen, Ching Monica; Yang, Hongyu; Xiang, Ye; Zhu, Guang

doi:10.1016/j.isci.2022.104976

Cited by 7 publications

(7 citation statements)

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“…In budding yeast, yMCM2-7 and yCdt1 form a complex, which stabilizes the spiral-shaped MCM2-7 complex 14 . In contrast, hMCM2-7 purified from human cells does not co-purify with hCdt1 34 . However, hCdt1 was found to interact with hMcm6 in pulldown assays 62 .…”

Section: The Role Of Hcdt1 In Hmcm2-7 Recruitmentmentioning

confidence: 86%

“…Whether the conformational flexibility of hORC has a functional or regulatory role in pre-RC formation is unknown. hMCM2-7 forms a stable complex in solution that adopts a spiral configuration similar to yMCM2-7, but unlike the yeast complex, does not co-purify with hCdt1 23,34 . Thus, how hCdt1 becomes recruited during human DNA licensing is not entirely clear.…”

Section: Introductionmentioning

confidence: 99%

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Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Wells,

Leber,

Edwardes

et al. 2024

Preprint

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Human DNA licensing initiates the process of replication fork assembly. Specifically, this reaction leads to the loading of hMCM2-7 on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the biochemical reconstitution of human DNA licensing using purified proteins, the structural and functional analysis of the process and reveal the impact of cancer-associated mutations on DNA licensing. We showed that the in vitro reaction is specific and results in the assembly of high-salt resistant hMCM2-7 double-hexamers, the final product of DNA licensing. We used ATPγS to block complex assembly at the hOrc1-5-Cdc6-Cdt1-MCM2-7 step. We observed that the assembly of this intermediate is independent of hOrc6, although hOrc6 enhances the loading of the second hMCM2-7 hexamer. The structural and mutational analysis of the hOrc1-5-Cdc6-Cdt1-MCM2 7 complex provides insights into hORC-Cdc6 dependent recruitment of hMCM2-7 via five hMcm winged-helix domains. The structure highlights how hOrc1 activates the hCdc6 ATPase, while the analysis of hOrc1 and hCdc6 ATPase mutants uncovered an unexpected role for hCdc6 ATPase in complex disassembly. The structure highlights that Cdc6 binding to Orc1-5 stabilises Orc2-DNA interactions and supports Mcm3-dependent recruitment of MCM2-7. Finally, the structure allowed us to locate cancer-associated mutations at the hCdc6-Mcm3 interface, which showed specific helicase loading defects.

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Section: The Role Of Hcdt1 In Hmcm2-7 Recruitmentmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Wells,

Leber,

Edwardes

et al. 2024

Preprint

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“…5a-b ). MCM self-association has been reported for endogenous human MCM2-7, but the nature of the complex formed had been unclear 69 . While there is currently no evidence to support that these MCM dimers can be directly loaded onto DNA, we speculate that the Mcm5/3/7 inter-hexamer interface observed in the dimer may represent the initial contact during double hexamer formation.…”

Section: Discussionmentioning

confidence: 99%

“…5a-b). MCM self-association has been reported for endogenous human MCM2-7, but the nature of the complex formed had been unclear 69 .…”

Section: Discussionmentioning

confidence: 99%

Multiple pathways for licensing human replication origins

Yang,

Hunker,

Wise

et al. 2024

Preprint

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SummaryThe loading of replicative helicases constitutes an obligatory step in the assembly of DNA replication machineries. In eukaryotes, the MCM2-7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head MCM double hexamer to license replication origins. Although extensively studied in the budding yeast model system, the mechanisms of origin licensing in higher eukaryotes remain poorly defined. Here, we use biochemical reconstitution and electron microscopy (EM) to reconstruct the human MCM loading pathway. Unexpectedly, we find that, unlike in yeast, ORC’s Orc6 subunit is not essential for human MCM loading but can enhance loading efficiency. EM analyses identify several intermediatesen routeto MCM double hexamer formation in the presence and absence of Orc6, including an abundant DNA-loaded, closed-ring single MCM hexamer intermediate that can mature into a head-to-head double hexamer through different pathways. In an Orc6-facilitated pathway, ORC and a second MCM2-7 hexamer are recruited to the dimerization interface of the first hexamer through an MCM-ORC intermediate that is architecturally distinct from an analogous intermediate in yeast. In an alternative, Orc6-independent pathway, MCM double hexamer formation proceeds through dimerization of two independently loaded single MCM2-7 hexamers, promoted by a propensity of human MCM2-7 hexamers to dimerize without the help of other loading factors. This redundancy in human MCM loading pathways likely provides resilience against replication stress under cellular conditions by ensuring that enough origins are licensed for efficient DNA replication. Additionally, the biochemical reconstitution of human origin licensing paves the way to address many outstanding questions regarding DNA replication initiation and replication-coupled events in higher eukaryotes in the future.

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“…These complexes are often challenging to study using other structural biology techniques such as X-ray crystallography, due to difficulties in obtaining large, well-ordered crystals. Cryo-EM can overcome these challenges by allowing for the visualization of large, flexible, and heterogeneous complexes in solution. , This is especially true for multisubunit nucleic-acid processing complexes including RNA polymerases, − DNA polymerases, − helicases, ,, and spliceosomes. , Sample preparation and grid-making remain the most challenging aspects of the cryo-EM pipeline (recently reviewed in ref ). Despite intensive research and technological advances, predicting which samples can be imaged by single-particle cryo-EM is still difficult and an area of active research.…”

Section: Technological Advancesmentioning

confidence: 99%

Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines

Lewis,

van Oijen,

Spenkelink

2023

Chem. Rev.

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The paradigm of cellular systems as deterministic machines has long guided our understanding of biology. Advancements in technology and methodology, however, have revealed a world of stochasticity, challenging the notion of determinism. Here, we explore the stochastic behavior of multi-protein complexes, using the DNA replication system (replisome) as a prime example. The faithful and timely copying of DNA depends on the simultaneous action of a large set of enzymes and scaffolding factors. This fundamental cellular process is underpinned by dynamic protein–nucleic acid assemblies that must transition between distinct conformations and compositional states. Traditionally viewed as a well-orchestrated molecular machine, recent experimental evidence has unveiled significant variability and heterogeneity in the replication process. In this review, we discuss recent advances in single-molecule approaches and single-particle cryo-EM, which have provided insights into the dynamic processes of DNA replication. We comment on the new challenges faced by structural biologists and biophysicists as they attempt to describe the dynamic cascade of events leading to replisome assembly, activation, and progression. The fundamental principles uncovered and yet to be discovered through the study of DNA replication will inform on similar operating principles for other multi-protein complexes.

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Cryo-EM structure of human hexameric MCM2-7 complex

Cited by 7 publications

References 50 publications

Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Multiple pathways for licensing human replication origins

Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines

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