Structure of a human replisome shows the organisation and interactions of a DNA replication machine

Jones, Morgan L.; Baris, Yasemin; Taylor, Martin R.G.; Yeeles, Joseph T.P.

doi:10.15252/embj.2021108819

Cited by 93 publications

(92 citation statements)

References 95 publications

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“…Ubiquitylation of CMG by both SCF Dia2 and CUL2 LRR1 is suppressed by the excluded DNA strand at replication forks [8][9][10] ; our discovery that Dia2 and LRR1 bind directly to a common site across the ZnF domains of MCM3 and MCM5 suggested that this region of MCM might be important for the regulation of ubiquitylation. In our recent structure of the human replisome bound to a replication fork 28 , cryo-EM density that we attributed to the excluded strand was positioned in the channel between the ZnF domains of MCM3 and MCM5, consistent with previous structures of Drosophila and budding yeast CMG 13,31,32 . To further validate our assignment of the excluded strand, we identified a subset of particles lacking CUL2 LRR1 from our dataset of replisomes assembled without an excluded strand (Extended Data Figs.…”

Section: Regulation Of Cmg Ubiquitylationsupporting

confidence: 88%

“…8, 9g). In the resulting density map, the MCM N-tier was identical to our previous map of replication fork-associated CMG 28 , apart from a single region of density, extending from the fork junction between the ZnF domains of MCM3 and MCM5 (Fig. 4a, Extended Data Fig.…”

Section: Regulation Of Cmg Ubiquitylationsupporting

confidence: 67%

“…The overall architecture of human CMG, AND-1, TIMELESS-TIPIN and Pol-ε are indistinguishable from our previous structure lacking CUL2 LRR1 (ref. 28 ) (Fig. 3a, b, Extended Data Fig.…”

Section: Human Replisome-cul2 Lrr1 Structurementioning

confidence: 96%

“…Although LRR1 displays no apparent sequence homology to Dia2, metazoan CUL2 LRR1 ubiquitylates CMG on its MCM7 subunit [4][5][6] and is suppressed by the excluded DNA strand 9,10 , suggesting there might be common features of replisome association that are important for the regulation of both SCF Dia2 and CUL2 LRR1 . To investigate this, we used our approach for human replisome assembly 28 and a DNA substrate lacking a 5′ flap, to determine a high-resolution structure of CUL2 LRR1 in the human replisome (Fig. 3a, b, Extended Data Figs.…”

Section: Human Replisome-cul2 Lrr1 Structurementioning

confidence: 99%

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A conserved mechanism for regulating replisome disassembly in eukaryotes

Jenkyn-Bedford

Jones

Baris

et al. 2021

Nature

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Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase1–3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4–7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8–10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome–E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR–MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

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Section: Regulation Of Cmg Ubiquitylationsupporting

confidence: 88%

Section: Regulation Of Cmg Ubiquitylationsupporting

confidence: 67%

“…The overall architecture of human CMG, AND-1, TIMELESS-TIPIN and Pol-ε are indistinguishable from our previous structure lacking CUL2 LRR1 (ref. 28 ) (Fig. 3a, b, Extended Data Fig.…”

Section: Human Replisome-cul2 Lrr1 Structurementioning

confidence: 96%

Section: Human Replisome-cul2 Lrr1 Structurementioning

confidence: 99%

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A conserved mechanism for regulating replisome disassembly in eukaryotes

Jenkyn-Bedford

Jones

Baris

et al. 2021

Nature

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“…We therefore propose that POLE2 is critical at one of the later steps of replication initiation, possibly at the structural perturbations associated with the polymerase switch. According to a recent structural study (38), human POLE2 binds the GINS-MCM junction of the CMG helicase, which could make POLE2 essential for the CMG stability during some conformational changes, such as the polymerase switch step. Further biochemical and structural studied are necessary to address this question.…”

Section: Discussionmentioning

confidence: 99%

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells

Vipat

Gupta

Jonchhe

et al. 2022

Preprint

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DNA polymerase epsilon in an essential enzyme, responsible for the synthesis of the leading strand during DNA replication. Deficiencies and mutations in DNA polymerase epsilon catalytic subunit (POLE1) cause severe developmental abnormalities and cancers. Paradoxically, the non-catalytic C-terminal domain of yeast polymerase epsilon catalytic subunit (Pol2) is sufficient for cell survival. The non-catalytic essential function of Pol2 in yeast has been associated with its role in the assembly of the replicative helicase CMG. However, the understanding of POLE1 functions in DNA replication initiation in human cells is falling behind. In this study we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in human cells. Surprisingly, in the absence of POLE1, human cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 C-terminal non-catalytic domain was enough to rescue replication initiation and support slow, but processive DNA synthesis, which was dependent on the POLE1-POLE2 interaction. We propose a model where in human cells POLE1/POLE2 are not essential for CMG assembly, but are required during later steps of replication initiation. Our study provides some insights into the role of DNA polymerase epsilon in replication initiation in human cells.

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DNA replication: Mechanisms and therapeutic interventions for diseases

Song

Shen

Mahasin

et al. 2023

MedComm

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Accurate and integral cellular DNA replication is modulated by multiple replication-associated proteins, which is fundamental to preserve genome stability. Furthermore, replication proteins cooperate with multiple DNA damage factors to deal with replication stress through mechanisms beyond their role in replication. Cancer cells with chronic replication stress exhibit aberrant DNA replication and DNA damage response, providing an exploitable therapeutic target in tumors. Numerous evidence has indicated that posttranslational modifications (PTMs) of replication proteins present distinct functions in DNA replication and respond to replication stress. In addition, abundant replication proteins are involved in tumorigenesis and development, which act as diagnostic and prognostic biomarkers in some tumors, implying these proteins act as therapeutic targets in clinical. Replication-target cancer therapy emerges as the times require. In this context, we outline the current investigation of the DNA replication mechanism, and simultaneously enumerate the aberrant expression of replication proteins as hallmark for various diseases, revealing their therapeutic potential for target therapy. Meanwhile, we also discuss current observations that the novel PTM of replication proteins in response to replication stress, which seems to be a promising strategy to eliminate diseases.

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Structure of a human replisome shows the organisation and interactions of a DNA replication machine

Cited by 93 publications

References 95 publications

A conserved mechanism for regulating replisome disassembly in eukaryotes

A conserved mechanism for regulating replisome disassembly in eukaryotes

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells

DNA replication: Mechanisms and therapeutic interventions for diseases

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