1
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Inès D, Leray A, Winckler P, Courty PE, Wendehenne D, Rosnoblet C. The nuclear dynamic of CDC48 is affected during the immune response in plants. PLANT SIGNALING & BEHAVIOR 2025; 20:2488104. [PMID: 40169277 PMCID: PMC11970785 DOI: 10.1080/15592324.2025.2488104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/07/2025] [Revised: 03/26/2025] [Accepted: 03/29/2025] [Indexed: 04/03/2025]
Abstract
Plants are continuously challenged by a myriad of pathogenic microorganisms, including bacteria, viruses, fungi and oomycetes, against which they must defend themselves. The protein Cell Division Cycle 48 (CDC48), a key player of ubiquitin-proteasome system which segregates and remodels ubiquitinated proteins for degradation, is known to be mobilized during plant immunity. Moreover, the characterization of the nuclear role of CDC48 is of interest, in particular its regulation in nuclear processes such as chromatin remodeling, DNA repair and gene expression. In this regard, its nuclear functions during plant immunity remain underexplored. This study investigates the dynamics of CDC48 during plant immune responses. The biophysical analysis using the Fluorescence Correlation Spectroscopy (FCS) on tobacco leaves stably overexpressing GFP-CDC48 revealed that the nuclear dynamics of CDC48 changed after treatment with cryptogein, an elicitor of immune responses. FCS analysis revealed an increase of the nuclear mobility of CDC48 and a faster interaction of CDC48 with a wide range of nuclear partners shortly after cryptogein treatment. Overall, our study shows a nuclear regulation of the interaction of CDC48 with its potential partners and sheds new light on potential nuclear involvements of CDC48 following the triggering of defense mechanisms.
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Affiliation(s)
- Damien Inès
- Université Bourgogne Europe, Institut Agro Dijon, INRAE, UMR Agroécologie, Dijon, France
| | - Aymeric Leray
- Laboratoire Interdisciplinaire Carnot de Bourgogne CNRS UMR 6303, Université Bourgogne Europe, Dijon, France
| | - Pascale Winckler
- Dimacell Imaging Facility, L’Institut Agro Dijon, INRAe, INSERM, Université Bourgogne Europe, Université Marie & Louis Pasteur, Dijon, France
| | - Pierre-Emmanuel Courty
- Université Bourgogne Europe, Institut Agro Dijon, INRAE, UMR Agroécologie, Dijon, France
| | - David Wendehenne
- Université Bourgogne Europe, Institut Agro Dijon, INRAE, UMR Agroécologie, Dijon, France
| | - Claire Rosnoblet
- Université Bourgogne Europe, Institut Agro Dijon, INRAE, UMR Agroécologie, Dijon, France
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2
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Kiss L, James LC, Schulman BA. UbiREAD deciphers proteasomal degradation code of homotypic and branched K48 and K63 ubiquitin chains. Mol Cell 2025; 85:1467-1476.e6. [PMID: 40132582 DOI: 10.1016/j.molcel.2025.02.021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Revised: 11/13/2024] [Accepted: 02/25/2025] [Indexed: 03/27/2025]
Abstract
Ubiquitin chains define the fates of their modified proteins, often mediating proteasomal degradation in eukaryotes. Yet heterogeneity of intracellular ubiquitination has precluded systematically comparing the degradation capacities of different ubiquitin chains. We developed ubiquitinated reporter evaluation after intracellular delivery (UbiREAD), a technology that monitors cellular degradation and deubiquitination at high temporal resolution after bespoke ubiquitinated proteins are delivered into human cells. Comparing the degradation of a model substrate modified with various K48, K63, or K48/K63-branched ubiquitin chains revealed fundamental differences in their intracellular degradation capacities. K48 chains with three or more ubiquitins triggered degradation within minutes. K63-ubiquitinated substrate was rapidly deubiquitinated rather than degraded. Surprisingly, in K48/K63-branched chains, substrate-anchored chain identity determined the degradation and deubiquitination behavior, establishing that branched chains are not the sum of their parts. UbiREAD reveals a degradation code for ubiquitin chains varying by linkage, length, and topology and a functional hierarchy within branched ubiquitin chains.
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Affiliation(s)
- Leo Kiss
- Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
| | - Leo C James
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - Brenda A Schulman
- Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried 82152, Germany
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3
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Shiomi Y, Hayashi A, Saito Y, Kanemaki MT, Nishitani H. The Depletion of TRAIP Results in the Retention of PCNA on Chromatin During Mitosis Leads to Inhibiting DNA Replication Initiation. Genes Cells 2025; 30:e70006. [PMID: 39956965 DOI: 10.1111/gtc.70006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2024] [Revised: 01/17/2025] [Accepted: 01/29/2025] [Indexed: 02/18/2025]
Abstract
Loading PCNA onto chromatin is a pivotal step in DNA replication, cell cycle progression, and genome integrity. Conversely, unloading PCNA from chromatin is equally crucial for maintaining genome stability. Cells deficient in the PCNA unloader ATAD5-RFC exhibit elevated levels of chromatin-bound PCNA during S phase, but still show dissociation of PCNA from chromatin in mitosis. In this study, we found that depletion of TRAIP, an E3 ubiquitin ligase, results in the retention of PCNA on chromatin during mitosis. Although TRAIP-depleted cells with chromatin-bound PCNA during mitosis progressed into the subsequent G1 phase, they displayed reduced levels of Cdt1, a key replication licensing factor, and impaired S phase entry. In addition, TRAIP-depleted cells exhibited delayed S phase progression. These results suggest that TRAIP functions independently of ATAD5-RFC in removing PCNA from chromatin. Furthermore, TRAIP appears to be essential for precise pre-replication complexes (pre-RCs) formation necessary for faithful initiation of DNA replication and S phase progression.
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Affiliation(s)
- Yasushi Shiomi
- Graduate School of Science, University of Hyogo, Kamigori, Japan
| | - Akiyo Hayashi
- Graduate School of Science, University of Hyogo, Kamigori, Japan
| | - Yuichiro Saito
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
| | - Masato T Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Shizuoka, Japan
- Department of Advanced Studies, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan
- Department of Biological Science, The University of Tokyo, Tokyo, Japan
| | - Hideo Nishitani
- Graduate School of Science, University of Hyogo, Kamigori, Japan
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4
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Xu Y, Morrow CA, Laksir Y, Holt OM, Taylor K, Tsiappourdhi C, Collins P, Jia S, Andreadis C, Whitby MC. DNA nicks in both leading and lagging strand templates can trigger break-induced replication. Mol Cell 2025; 85:91-106.e5. [PMID: 39561776 DOI: 10.1016/j.molcel.2024.10.026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 09/09/2024] [Accepted: 10/22/2024] [Indexed: 11/21/2024]
Abstract
Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted.
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Affiliation(s)
- Yuanlin Xu
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Carl A Morrow
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Yassine Laksir
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Orla M Holt
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Kezia Taylor
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Costas Tsiappourdhi
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Patrick Collins
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Su Jia
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Christos Andreadis
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Matthew C Whitby
- Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
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5
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Ahmed SMQ, Sasikumar J, Laha S, Das SP. Multifaceted role of the DNA replication protein MCM10 in maintaining genome stability and its implication in human diseases. Cancer Metastasis Rev 2024; 43:1353-1371. [PMID: 39240414 DOI: 10.1007/s10555-024-10209-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 08/29/2024] [Indexed: 09/07/2024]
Abstract
MCM10 plays a vital role in genome duplication and is crucial for DNA replication initiation, elongation, and termination. It coordinates several proteins to assemble at the fork, form a functional replisome, trigger origin unwinding, and stabilize the replication bubble. MCM10 overexpression is associated with increased aggressiveness in breast, cervical, and several other cancers. Disruption of MCM10 leads to altered replication timing associated with initiation site gains and losses accompanied by genome instability. Knockdown of MCM10 affects the proliferation and migration of cancer cells, manifested by DNA damage and replication fork arrest, and has recently been shown to be associated with clinical conditions like CNKD and RCM. Loss of MCM10 function is associated with impaired telomerase activity, leading to the accumulation of abnormal replication forks and compromised telomere length. MCM10 interacts with histones, aids in nucleosome assembly, binds BRCA2 to maintain genome integrity during DNA damage, prevents lesion skipping, and inhibits PRIMPOL-mediated repriming. It also interacts with the fork reversal enzyme SMARCAL1 and inhibits fork regression. Additionally, MCM10 undergoes several post-translational modifications and contributes to transcriptional silencing by interacting with the SIR proteins. This review explores the mechanism associated with MCM10's multifaceted role in DNA replication initiation, chromatin organization, transcriptional silencing, replication stress, fork stability, telomere length maintenance, and DNA damage response. Finally, we discuss the role of MCM10 in the early detection of cancer, its prognostic significance, and its potential use in therapeutics for cancer treatment.
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Affiliation(s)
- Sumayyah M Q Ahmed
- Cell Biology and Molecular Genetics (CBMG), Yenepoya Research Centre (YRC), Yenepoya (Deemed to be University), Mangalore, 575018, India
| | - Jayaprakash Sasikumar
- Cell Biology and Molecular Genetics (CBMG), Yenepoya Research Centre (YRC), Yenepoya (Deemed to be University), Mangalore, 575018, India
| | - Suparna Laha
- Cell Biology and Molecular Genetics (CBMG), Yenepoya Research Centre (YRC), Yenepoya (Deemed to be University), Mangalore, 575018, India
| | - Shankar Prasad Das
- Cell Biology and Molecular Genetics (CBMG), Yenepoya Research Centre (YRC), Yenepoya (Deemed to be University), Mangalore, 575018, India.
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6
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Can G, Shyian M, Krishnamoorthy A, Lim Y, Wu RA, Zaher MS, Raschle M, Walter JC, Pellman DS. TTF2 promotes replisome eviction from stalled forks in mitosis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.30.626186. [PMID: 39651145 PMCID: PMC11623681 DOI: 10.1101/2024.11.30.626186] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2024]
Abstract
When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.
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Affiliation(s)
- Geylani Can
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Maksym Shyian
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Archana Krishnamoorthy
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Yang Lim
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - R. Alex Wu
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Present Address: Arrakis Pharmaceuticals, Waltham, MA, USA
| | - Manal S. Zaher
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
| | - Markus Raschle
- Technische Universitat Kaiserslautern, Kaiserslautern, Germany
| | - Johannes C. Walter
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Howard Hughes Medical Institute
| | - David S. Pellman
- Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Howard Hughes Medical Institute
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7
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Olson O, Pelliciari S, Heron ED, Deegan TD. A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome. EMBO J 2024; 43:3846-3875. [PMID: 39039288 PMCID: PMC11405395 DOI: 10.1038/s44318-024-00168-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Revised: 06/24/2024] [Accepted: 06/26/2024] [Indexed: 07/24/2024] Open
Abstract
The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these 'accessory' helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome.
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Affiliation(s)
- Ottavia Olson
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK
| | - Simone Pelliciari
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK
| | - Emma D Heron
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK
| | - Tom D Deegan
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK.
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8
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Polo Rivera C, Deegan TD, Labib KPM. CMG helicase disassembly is essential and driven by two pathways in budding yeast. EMBO J 2024; 43:3818-3845. [PMID: 39039287 PMCID: PMC11405719 DOI: 10.1038/s44318-024-00161-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2024] [Revised: 06/12/2024] [Accepted: 06/19/2024] [Indexed: 07/24/2024] Open
Abstract
The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.
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Affiliation(s)
- Cristian Polo Rivera
- MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK
| | - Tom D Deegan
- MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, EH4 2XU, UK.
| | - Karim P M Labib
- MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
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9
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Atemin A, Ivanova A, Kanev PB, Uzunova S, Nedelcheva-Veleva M, Stoynov S. Dynamics of Replication-Associated Protein Levels through the Cell Cycle. Int J Mol Sci 2024; 25:8230. [PMID: 39125800 PMCID: PMC11311332 DOI: 10.3390/ijms25158230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 07/25/2024] [Accepted: 07/25/2024] [Indexed: 08/12/2024] Open
Abstract
The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.
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Affiliation(s)
| | | | | | | | | | - Stoyno Stoynov
- Laboratory of Genomic Stability, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G., Bonchev Str. Bl. 21, 1113 Sofia, Bulgaria; (A.A.); (A.I.); (P.-B.K.); (S.U.); (M.N.-V.)
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10
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Jones RM, Ruiz JH, Scaramuzza S, Nath S, Liu C, Henklewska M, Natsume T, Bristow RG, Romero F, Kanemaki MT, Gambus A. Characterizing replisome disassembly in human cells. iScience 2024; 27:110260. [PMID: 39055910 PMCID: PMC11269944 DOI: 10.1016/j.isci.2024.110260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 03/22/2024] [Accepted: 06/10/2024] [Indexed: 07/28/2024] Open
Abstract
To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.
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Affiliation(s)
- Rebecca M. Jones
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Joaquin Herrero Ruiz
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Shaun Scaramuzza
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Sarmi Nath
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Chaoyu Liu
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Marta Henklewska
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Toyoaki Natsume
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Robert G. Bristow
- Cancer Research UK – Manchester Institute, Manchester Cancer Research Center, Manchester, UK
| | - Francisco Romero
- Department of Microbiology, University of Seville, Seville, Spain
| | - Masato T. Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
- Department of Biological Science, The University of Tokyo, Tokyo, Japan
| | - Agnieszka Gambus
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
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11
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Noireterre A, Stutz F. Cdc48/p97 segregase: Spotlight on DNA-protein crosslinks. DNA Repair (Amst) 2024; 139:103691. [PMID: 38744091 DOI: 10.1016/j.dnarep.2024.103691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Revised: 04/30/2024] [Accepted: 05/01/2024] [Indexed: 05/16/2024]
Abstract
The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs. This review introduces the detailed structure, mechanism and cellular functions of Cdc48/p97 with an emphasis on the current knowledge of DNA-protein crosslink repair pathways across several organisms. The review concludes by discussing the potential therapeutic relevance of targeting p97 in DPC repair.
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Affiliation(s)
- Audrey Noireterre
- Department of Molecular and Cellular Biology, University of Geneva, Geneva 4 1211, Switzerland
| | - Françoise Stutz
- Department of Molecular and Cellular Biology, University of Geneva, Geneva 4 1211, Switzerland.
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12
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Jurkovic CM, Raisch J, Tran S, Nguyen HD, Lévesque D, Scott MS, Campos EI, Boisvert FM. Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks. Mol Cell Proteomics 2024; 23:100767. [PMID: 38615877 PMCID: PMC11101681 DOI: 10.1016/j.mcpro.2024.100767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Revised: 03/19/2024] [Accepted: 04/11/2024] [Indexed: 04/16/2024] Open
Abstract
DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.
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Affiliation(s)
- Carla-Marie Jurkovic
- Faculty of Medicine and Health Sciences, Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Jennifer Raisch
- Faculty of Medicine and Health Sciences, Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Stephanie Tran
- Genetics & Genome Biology Program, Department of Molecular Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
| | - Hoang Dong Nguyen
- Faculty of Medicine and Health Sciences, Department of Biochemistry and Functional Genomics, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Dominique Lévesque
- Faculty of Medicine and Health Sciences, Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Michelle S Scott
- Faculty of Medicine and Health Sciences, Department of Biochemistry and Functional Genomics, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Eric I Campos
- Genetics & Genome Biology Program, Department of Molecular Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.
| | - François-Michel Boisvert
- Faculty of Medicine and Health Sciences, Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada.
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13
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Jurkovic CM, Boisvert FM. Evolution of techniques and tools for replication fork proteome and protein interaction studies. Biochem Cell Biol 2024; 102:135-144. [PMID: 38113480 DOI: 10.1139/bcb-2023-0215] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2023] Open
Abstract
Understanding the complex network of protein-protein interactions (PPI) that govern cellular functions is essential for unraveling the molecular basis of biological processes and diseases. Mass spectrometry (MS) has emerged as a powerful tool for studying protein dynamics, enabling comprehensive analysis of protein function, structure, post-translational modifications, interactions, and localization. This article provides an overview of MS techniques and their applications in proteomics studies, with a focus on the replication fork proteome. The replication fork is a multi-protein assembly involved in DNA replication, and its proper functioning is crucial for maintaining genomic integrity. By combining quantitative MS labeling techniques with various data acquisition methods, researchers have made significant strides in elucidating the complex processes and molecular mechanisms at the replication fork. Overall, MS has revolutionized our understanding of protein dynamics, offering valuable insights into cellular processes and potential targets for therapeutic interventions.
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Affiliation(s)
- Carla-Marie Jurkovic
- Department of Immunology and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
| | - François-Michel Boisvert
- Department of Immunology and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
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14
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Jones RM, Reynolds-Winczura A, Gambus A. A Decade of Discovery-Eukaryotic Replisome Disassembly at Replication Termination. BIOLOGY 2024; 13:233. [PMID: 38666845 PMCID: PMC11048390 DOI: 10.3390/biology13040233] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2024] [Revised: 03/23/2024] [Accepted: 03/25/2024] [Indexed: 04/28/2024]
Abstract
The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations.
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Affiliation(s)
- Rebecca M. Jones
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham B15 2TT, UK; (R.M.J.); (A.R.-W.)
- School of Biosciences, Aston University, Birmingham B4 7ET, UK
| | - Alicja Reynolds-Winczura
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham B15 2TT, UK; (R.M.J.); (A.R.-W.)
| | - Agnieszka Gambus
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham B15 2TT, UK; (R.M.J.); (A.R.-W.)
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15
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Kumar C, Remus D. Looping out of control: R-loops in transcription-replication conflict. Chromosoma 2024; 133:37-56. [PMID: 37419963 PMCID: PMC10771546 DOI: 10.1007/s00412-023-00804-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/27/2023] [Accepted: 06/28/2023] [Indexed: 07/09/2023]
Abstract
Transcription-replication conflict is a major cause of replication stress that arises when replication forks collide with the transcription machinery. Replication fork stalling at sites of transcription compromises chromosome replication fidelity and can induce DNA damage with potentially deleterious consequences for genome stability and organismal health. The block to DNA replication by the transcription machinery is complex and can involve stalled or elongating RNA polymerases, promoter-bound transcription factor complexes, or DNA topology constraints. In addition, studies over the past two decades have identified co-transcriptional R-loops as a major source for impairment of DNA replication forks at active genes. However, how R-loops impede DNA replication at the molecular level is incompletely understood. Current evidence suggests that RNA:DNA hybrids, DNA secondary structures, stalled RNA polymerases, and condensed chromatin states associated with R-loops contribute to the of fork progression. Moreover, since both R-loops and replication forks are intrinsically asymmetric structures, the outcome of R-loop-replisome collisions is influenced by collision orientation. Collectively, the data suggest that the impact of R-loops on DNA replication is highly dependent on their specific structural composition. Here, we will summarize our current understanding of the molecular basis for R-loop-induced replication fork progression defects.
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Affiliation(s)
- Charanya Kumar
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, 10065, USA
| | - Dirk Remus
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, 10065, USA.
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16
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Lewis JS, van Oijen AM, Spenkelink LM. Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines. Chem Rev 2023; 123:13419-13440. [PMID: 37971892 PMCID: PMC10790245 DOI: 10.1021/acs.chemrev.3c00436] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 10/15/2023] [Accepted: 10/20/2023] [Indexed: 11/19/2023]
Abstract
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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Affiliation(s)
- Jacob S. Lewis
- Macromolecular
Machines Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Antoine M. van Oijen
- Molecular
Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales 2522, Australia
| | - Lisanne M. Spenkelink
- Molecular
Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales 2522, Australia
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17
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Xia Y, Sonneville R, Jenkyn-Bedford M, Ji L, Alabert C, Hong Y, Yeeles JT, Labib KP. DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in Caenorhabditis elegans. Science 2023; 381:eadi4932. [PMID: 37590372 PMCID: PMC7615117 DOI: 10.1126/science.adi4932] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Accepted: 08/07/2023] [Indexed: 08/19/2023]
Abstract
Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.
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Affiliation(s)
- Yisui Xia
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, U.K
| | - Remi Sonneville
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, U.K
| | | | - Liqin Ji
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Constance Alabert
- Division of Molecular, Cell & Developmental Biology, School of Life Sciences, University of Dundee, Dundee, U.K
| | - Ye Hong
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, U.K
- Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao, China
| | - Joseph T.P. Yeeles
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, U.K
| | - Karim P.M. Labib
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, U.K
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18
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Varshavsky A, Lewis K, Chen SJ. Deletions of DNA in cancer and their possible uses for therapy. Bioessays 2023; 45:e2300051. [PMID: 37166062 PMCID: PMC11102808 DOI: 10.1002/bies.202300051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2023] [Revised: 04/09/2023] [Accepted: 04/12/2023] [Indexed: 05/12/2023]
Abstract
Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed "replication blocks specific for deletions" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.
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Affiliation(s)
- Alexander Varshavsky
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA
| | - Kim Lewis
- Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, Massachusetts, USA
| | - Shun-Jia Chen
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA
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19
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Shrestha S, Minamino M, Chen ZA, Bouchoux C, Rappsilber J, Uhlmann F. Replisome-cohesin interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors. Chromosoma 2023; 132:117-135. [PMID: 37166686 PMCID: PMC10247859 DOI: 10.1007/s00412-023-00797-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Revised: 04/25/2023] [Accepted: 04/26/2023] [Indexed: 05/12/2023]
Abstract
The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.
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Affiliation(s)
- Sudikchya Shrestha
- Chromosome Segregation Laboratory, The Francis Crick Institute, London, NW1 1AT, UK
| | - Masashi Minamino
- Chromosome Segregation Laboratory, The Francis Crick Institute, London, NW1 1AT, UK
| | - Zhuo A Chen
- Bioanalytics Unit, Institute of Biotechnology, Technische Universität Berlin, 13355, Berlin, Germany
| | - Céline Bouchoux
- Chromosome Segregation Laboratory, The Francis Crick Institute, London, NW1 1AT, UK
| | - Juri Rappsilber
- Bioanalytics Unit, Institute of Biotechnology, Technische Universität Berlin, 13355, Berlin, Germany
- Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3BF, UK
| | - Frank Uhlmann
- Chromosome Segregation Laboratory, The Francis Crick Institute, London, NW1 1AT, UK.
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20
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Zhou M, Fang R, Colson L, Donovan KA, Hunkeler M, Song Y, Zhang C, Chen S, Lee DH, Bradshaw GA, Eisert R, Ye Y, Kalocsay M, Goldberg A, Fischer ES, Lu Y. HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.30.542866. [PMID: 37398461 PMCID: PMC10312588 DOI: 10.1101/2023.05.30.542866] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/04/2023]
Abstract
Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.
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21
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Noireterre A, Serbyn N, Bagdiul I, Stutz F. Ubx5-Cdc48 assists the protease Wss1 at DNA-protein crosslink sites in yeast. EMBO J 2023:e113609. [PMID: 37144685 DOI: 10.15252/embj.2023113609] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 04/14/2023] [Accepted: 04/24/2023] [Indexed: 05/06/2023] Open
Abstract
DNA-protein crosslinks (DPCs) pose a serious threat to genome stability. The yeast proteases Wss1, 26S proteasome, and Ddi1 are safeguards of genome integrity by acting on a plethora of DNA-bound proteins in different cellular contexts. The AAA ATPase Cdc48/p97 is known to assist Wss1/SPRTN in clearing DNA-bound complexes; however, its contribution to DPC proteolysis remains unclear. Here, we show that the Cdc48 adaptor Ubx5 is detrimental in yeast mutants defective in DPC processing. Using an inducible site-specific crosslink, we show that Ubx5 accumulates at persistent DPC lesions in the absence of Wss1, which prevents their efficient removal from the DNA. Abolishing Cdc48 binding or complete loss of Ubx5 suppresses sensitivity of wss1∆ cells to DPC-inducing agents by favoring alternate repair pathways. We provide evidence for cooperation of Ubx5-Cdc48 and Wss1 in the genotoxin-induced degradation of RNA polymerase II (RNAPII), a described candidate substrate of Wss1. We propose that Ubx5-Cdc48 assists Wss1 for proteolysis of a subset of DNA-bound proteins. Together, our findings reveal a central role for Ubx5 in DPC clearance and repair.
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Affiliation(s)
- Audrey Noireterre
- Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland
| | - Nataliia Serbyn
- Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland
| | - Ivona Bagdiul
- Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland
| | - Françoise Stutz
- Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland
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22
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Jaffray EG, Tatham MH, Mojsa B, Liczmanska M, Rojas-Fernandez A, Yin Y, Ball G, Hay RT. The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA. J Cell Biol 2023; 222:e202201027. [PMID: 36880596 PMCID: PMC10005898 DOI: 10.1083/jcb.202201027] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 10/27/2022] [Accepted: 01/04/2023] [Indexed: 03/04/2023] Open
Abstract
Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.
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Affiliation(s)
- Ellis G. Jaffray
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Michael H. Tatham
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Barbara Mojsa
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Magda Liczmanska
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Alejandro Rojas-Fernandez
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Yili Yin
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Graeme Ball
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Ronald T. Hay
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
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23
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The CMG helicase and cancer: a tumor "engine" and weakness with missing mutations. Oncogene 2023; 42:473-490. [PMID: 36522488 PMCID: PMC9948756 DOI: 10.1038/s41388-022-02572-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 12/01/2022] [Accepted: 12/07/2022] [Indexed: 12/23/2022]
Abstract
The replicative Cdc45-MCM-GINS (CMG) helicase is a large protein complex that functions in the DNA melting and unwinding steps as a component of replisomes during DNA replication in mammalian cells. Although the CMG performs this important role in cell growth, the CMG is not a simple bystander in cell cycle events. Components of the CMG, specifically the MCM precursors, are also involved in maintaining genomic stability by regulating DNA replication fork speeds, facilitating recovery from replicative stresses, and preventing consequential DNA damage. Given these important functions, MCM/CMG complexes are highly regulated by growth factors such as TGF-ß1 and by signaling factors such as Myc, Cyclin E, and the retinoblastoma protein. Mismanagement of MCM/CMG complexes when these signaling mediators are deregulated, and in the absence of the tumor suppressor protein p53, leads to increased genomic instability and is a contributor to tumorigenic transformation and tumor heterogeneity. The goal of this review is to provide insight into the mechanisms and dynamics by which the CMG is regulated during its assembly and activation in mammalian genomes, and how errors in CMG regulation due to oncogenic changes promote tumorigenesis. Finally, and most importantly, we highlight the emerging understanding of the CMG helicase as an exploitable vulnerability and novel target for therapeutic intervention in cancer.
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24
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Song H, Shen R, Mahasin H, Guo Y, Wang D. DNA replication: Mechanisms and therapeutic interventions for diseases. MedComm (Beijing) 2023; 4:e210. [PMID: 36776764 PMCID: PMC9899494 DOI: 10.1002/mco2.210] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Revised: 01/08/2023] [Accepted: 01/09/2023] [Indexed: 02/09/2023] Open
Abstract
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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Affiliation(s)
- Hao‐Yun Song
- School of Basic Medical SciencesLanzhou UniversityLanzhouGansuChina
| | - Rong Shen
- School of Basic Medical SciencesLanzhou UniversityLanzhouGansuChina
| | - Hamid Mahasin
- School of Basic Medical SciencesLanzhou UniversityLanzhouGansuChina
| | - Ya‐Nan Guo
- School of Basic Medical SciencesLanzhou UniversityLanzhouGansuChina
| | - De‐Gui Wang
- School of Basic Medical SciencesLanzhou UniversityLanzhouGansuChina
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25
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Zhang W, Tang M, Wang L, Zhou H, Gao J, Chen Z, Zhao B, Zheng P. Lnc956-TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis. SCIENCE ADVANCES 2023; 9:eadf6277. [PMID: 36706191 PMCID: PMC9882984 DOI: 10.1126/sciadv.adf6277] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/02/2022] [Accepted: 12/22/2022] [Indexed: 06/18/2023]
Abstract
Replication stress is a major source of endogenous DNA damage. Despite the identification of numerous proteins on replication forks to modulate fork or replication machinery activities, it remains unexplored whether noncoding RNAs can localize on stalled forks and play critical regulatory roles. Here, we identify an uncharacterized long noncoding RNA NONMMUT028956 (Lnc956 for short) predominantly expressed in mouse embryonic stem cells. Lnc956 is accumulated on replication forks to prevent fork collapse and preserve genomic stability and is essential for mouse embryogenesis. Mechanistically, it drives assembly of the Lnc956-TRIM28-HSP90B1 complex on stalled forks in an interdependent manner downstream of ataxia telangiectasia and Rad3-related (ATR) signaling. Lnc956-TRIM28-HSP90B1 complex physically associates with minichromosome maintenance proteins 2 (MCM2) to minichromosome maintenance proteins 7 (MCM7) hexamer via TRIM28 and directly regulates the CDC45-MCM-GINS (CMG) helicase retention on chromatin. The regulation of Lnc956-TRIM28-HSP90B1 on CMG retention is mediated by HSP90B1's chaperoning function. These findings reveal a player that actively regulates replisome retention to prevent fork collapse.
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Affiliation(s)
- Weidao Zhang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
| | - Min Tang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- University of Chinese Academy of Sciences, Beijing 101408, China
| | - Lin Wang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
| | - Hu Zhou
- Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Jing Gao
- Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
| | - Zhongliang Chen
- Key Laboratory of Adult Stem Cell Translational Research (Chinese Academy of Medical Sciences), Guizhou Medical University, Guiyang, China
- National Joint Local Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Medical University, Guiyang, China
| | - Bo Zhao
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
| | - Ping Zheng
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- Key Laboratory of Animal Models and Human Disease Mechanisms of Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
- KIZ/CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
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26
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Kavlashvili T, Liu W, Mohamed TM, Cortez D, Dewar JM. Replication fork uncoupling causes nascent strand degradation and fork reversal. Nat Struct Mol Biol 2023; 30:115-124. [PMID: 36593312 PMCID: PMC9868089 DOI: 10.1038/s41594-022-00871-y] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 10/14/2022] [Indexed: 01/03/2023]
Abstract
Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and are exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts and validated this approach with experiments in human cells. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. Notably, the replicative helicase remains bound during NSD and fork reversal. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and elucidate key events that precede fork reversal.
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Affiliation(s)
- Tamar Kavlashvili
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Wenpeng Liu
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Taha M Mohamed
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - David Cortez
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - James M Dewar
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
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27
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Martín-Rufo R, de la Vega-Barranco G, Lecona E. Ubiquitin and SUMO as timers during DNA replication. Semin Cell Dev Biol 2022; 132:62-73. [PMID: 35210137 DOI: 10.1016/j.semcdb.2022.02.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2021] [Revised: 02/12/2022] [Accepted: 02/14/2022] [Indexed: 12/14/2022]
Abstract
Every time a cell copies its DNA the genetic material is exposed to the acquisition of mutations and genomic alterations that corrupt the information passed on to daughter cells. A tight temporal regulation of DNA replication is necessary to ensure the full copy of the DNA while preventing the appearance of genomic instability. Protein modification by ubiquitin and SUMO constitutes a very complex and versatile system that allows the coordinated control of protein stability, activity and interactome. In chromatin, their action is complemented by the AAA+ ATPase VCP/p97 that recognizes and removes ubiquitylated and SUMOylated factors from specific cellular compartments. The concerted action of the ubiquitin/SUMO system and VCP/p97 determines every step of DNA replication enforcing the ordered activation/inactivation, loading/unloading and stabilization/destabilization of replication factors. Here we analyze the mechanisms used by ubiquitin/SUMO and VCP/p97 to establish molecular timers throughout DNA replication and their relevance in maintaining genome stability. We propose that these PTMs are the main molecular watch of DNA replication from origin recognition to replisome disassembly.
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Affiliation(s)
- Rodrigo Martín-Rufo
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Guillermo de la Vega-Barranco
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Emilio Lecona
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain.
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28
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Vipat S, Gupta D, Jonchhe S, Anderspuk H, Rothenberg E, Moiseeva TN. The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells. Nat Commun 2022; 13:7099. [PMID: 36402816 PMCID: PMC9675812 DOI: 10.1038/s41467-022-34911-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Accepted: 11/10/2022] [Indexed: 11/21/2022] Open
Abstract
DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.
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Affiliation(s)
- Sameera Vipat
- Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, 12618, Estonia
| | - Dipika Gupta
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA
| | - Sagun Jonchhe
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA
| | - Hele Anderspuk
- Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, 12618, Estonia
| | - Eli Rothenberg
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA
| | - Tatiana N Moiseeva
- Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, 12618, Estonia.
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29
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Fujisawa R, Polo Rivera C, Labib KPM. Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase. eLife 2022; 11:e76763. [PMID: 35920641 PMCID: PMC9377798 DOI: 10.7554/elife.76763] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Accepted: 08/02/2022] [Indexed: 11/17/2022] Open
Abstract
The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.
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Affiliation(s)
- Ryo Fujisawa
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeDundeeUnited Kingdom
| | - Cristian Polo Rivera
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeDundeeUnited Kingdom
| | - Karim PM Labib
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeDundeeUnited Kingdom
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30
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Lewis JS, Gross MH, Sousa J, Henrikus SS, Greiwe JF, Nans A, Diffley JFX, Costa A. Mechanism of replication origin melting nucleated by CMG helicase assembly. Nature 2022; 606:1007-1014. [PMID: 35705812 PMCID: PMC9242855 DOI: 10.1038/s41586-022-04829-4] [Citation(s) in RCA: 58] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Accepted: 05/03/2022] [Indexed: 12/04/2022]
Abstract
The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.
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Affiliation(s)
- Jacob S Lewis
- Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK
| | - Marta H Gross
- Chromosome Replication Laboratory, The Francis Crick Institute, London, UK
| | - Joana Sousa
- Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK
- UCB Pharma, Slough, UK
| | - Sarah S Henrikus
- Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK
| | - Julia F Greiwe
- Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK
| | - Andrea Nans
- Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK
| | - John F X Diffley
- Chromosome Replication Laboratory, The Francis Crick Institute, London, UK
| | - Alessandro Costa
- Macromolecular Machines Laboratory, The Francis Crick Institute, London, UK.
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31
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Conwell SC, Cranford MT, Kavlashvili T, Dewar JM. Replication fork collapse in vitro using Xenopus egg extracts. Methods Enzymol 2022; 672:317-338. [DOI: 10.1016/bs.mie.2022.03.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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32
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Grabarczyk DB. The Fork Protection Complex: A Regulatory Hub at the Head of the Replisome. Subcell Biochem 2022; 99:83-107. [PMID: 36151374 DOI: 10.1007/978-3-031-00793-4_3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
As well as accurately duplicating DNA, the eukaryotic replisome performs a variety of other crucial tasks to maintain genomic stability. For example, organizational elements, like cohesin, must be transferred from the front of the fork to the new strands, and when there is replication stress, forks need to be protected and checkpoint signalling activated. The Tof1-Csm3 (or Timeless-Tipin in humans) Fork Protection Complex (FPC) ensures efficient replisome progression and is required for a range of replication-associated activities. Recent studies have begun to reveal the structure of this complex, and how it functions within the replisome to perform its diverse roles. The core of the FPC acts as a DNA grip on the front of the replisome to regulate fork progression. Other flexibly linked domains and motifs mediate interactions with proteins and specific DNA structures, enabling the FPC to act as a hub at the head of the replication fork.
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Affiliation(s)
- Daniel B Grabarczyk
- Rudolf Virchow Center for Integrative and Translational Bioimaging, Institute for Structural Biology, University of Würzburg, Würzburg, Germany.
- Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria.
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33
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Ivanova A, Atemin A, Uzunova S, Danovski G, Aleksandrov R, Stoynov S, Nedelcheva-Veleva M. The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae. Molecules 2021; 27:97. [PMID: 35011329 PMCID: PMC8746418 DOI: 10.3390/molecules27010097] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Revised: 12/21/2021] [Accepted: 12/22/2021] [Indexed: 01/13/2023] Open
Abstract
Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4-an essential DNA replication protein and substrate of Dia2-prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.
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Affiliation(s)
| | | | | | | | | | - Stoyno Stoynov
- Laboratory of Genomic Stability, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G., Bonchev Str. Bl.21, 1113 Sofia, Bulgaria; (A.I.); (A.A.); (S.U.); (G.D.); (R.A.)
| | - Marina Nedelcheva-Veleva
- Laboratory of Genomic Stability, Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G., Bonchev Str. Bl.21, 1113 Sofia, Bulgaria; (A.I.); (A.A.); (S.U.); (G.D.); (R.A.)
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34
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Zhou H, Zaher MS, Walter JC, Brown A. Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates. Nucleic Acids Res 2021; 49:13194-13206. [PMID: 34850944 PMCID: PMC8682755 DOI: 10.1093/nar/gkab1174] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 11/08/2021] [Accepted: 11/11/2021] [Indexed: 11/17/2022] Open
Abstract
When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1’s catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.
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Affiliation(s)
- Haixia Zhou
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Manal S Zaher
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
| | - Johannes C Walter
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.,Howard Hughes Medical Institute, Boston, MA 02115, USA
| | - Alan Brown
- Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
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35
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Jones ML, Baris Y, Taylor MRG, Yeeles JTP. Structure of a human replisome shows the organisation and interactions of a DNA replication machine. EMBO J 2021; 40:e108819. [PMID: 34694004 PMCID: PMC8634136 DOI: 10.15252/embj.2021108819] [Citation(s) in RCA: 79] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Revised: 09/28/2021] [Accepted: 09/30/2021] [Indexed: 02/01/2023] Open
Abstract
The human replisome is an elaborate arrangement of molecular machines responsible for accurate chromosome replication. At its heart is the CDC45-MCM-GINS (CMG) helicase, which, in addition to unwinding the parental DNA duplex, arranges many proteins including the leading-strand polymerase Pol ε, together with TIMELESS-TIPIN, CLASPIN and AND-1 that have key and varied roles in maintaining smooth replisome progression. How these proteins are coordinated in the human replisome is poorly understood. We have determined a 3.2 Å cryo-EM structure of a human replisome comprising CMG, Pol ε, TIMELESS-TIPIN, CLASPIN and AND-1 bound to replication fork DNA. The structure permits a detailed understanding of how AND-1, TIMELESS-TIPIN and Pol ε engage CMG, reveals how CLASPIN binds to multiple replisome components and identifies the position of the Pol ε catalytic domain. Furthermore, the intricate network of contacts contributed by MCM subunits and TIMELESS-TIPIN with replication fork DNA suggests a mechanism for strand separation.
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36
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Jenkyn-Bedford M, Jones ML, Baris Y, Labib KPM, Cannone G, Yeeles JTP, Deegan TD. A conserved mechanism for regulating replisome disassembly in eukaryotes. Nature 2021; 600:743-747. [PMID: 34700328 PMCID: PMC8695382 DOI: 10.1038/s41586-021-04145-3] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 10/14/2021] [Indexed: 11/28/2022]
Abstract
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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Affiliation(s)
| | | | | | - Karim P M Labib
- MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK
| | | | | | - Tom D Deegan
- MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK.
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh, UK.
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37
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Jameson KH, Rudolph CJ, Hawkins M. Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA. J Biol Chem 2021; 297:101409. [PMID: 34780717 DOI: 10.1016/j.jbc.2021.101409] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Revised: 11/02/2021] [Accepted: 11/10/2021] [Indexed: 02/05/2023] Open
Abstract
The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the 'replication fork trap' region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.
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Affiliation(s)
- Katie H Jameson
- Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK
| | - Christian J Rudolph
- Division of Biosciences, College of Health, Medicine and Life Sciences, Brunel University London, Uxbridge, UB8 3PH, UK
| | - Michelle Hawkins
- Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK.
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38
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Abdul Rehman SA, Armstrong LA, Lange SM, Kristariyanto YA, Gräwert TW, Knebel A, Svergun DI, Kulathu Y. Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2. Mol Cell 2021; 81:4176-4190.e6. [PMID: 34529927 PMCID: PMC8550791 DOI: 10.1016/j.molcel.2021.08.024] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 03/29/2021] [Accepted: 08/19/2021] [Indexed: 01/10/2023]
Abstract
Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.
The catalytic domain of MINDY1/2 has five distinct Ub binding sites Dynamics of the Cys loop regulate DUB activity Non-canonical catalytic triad composed of Cys-His-Thr MINDY1/2 uses an exo- or endo-cleavage mode that is determined by Ub chain length
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Affiliation(s)
- Syed Arif Abdul Rehman
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
| | - Lee A Armstrong
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
| | - Sven M Lange
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
| | - Yosua Adi Kristariyanto
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
| | - Tobias W Gräwert
- European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Notkestr. 85, 22607 Hamburg, Germany
| | - Axel Knebel
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
| | - Dmitri I Svergun
- European Molecular Biology Laboratory, Hamburg Unit, EMBL c/o DESY, Notkestr. 85, 22607 Hamburg, Germany
| | - Yogesh Kulathu
- MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
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39
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Kumar C, Batra S, Griffith JD, Remus D. The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions. eLife 2021; 10:72286. [PMID: 34494544 PMCID: PMC8479836 DOI: 10.7554/elife.72286] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2021] [Accepted: 09/07/2021] [Indexed: 12/11/2022] Open
Abstract
R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.
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Affiliation(s)
- Charanya Kumar
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
| | - Sahil Batra
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
| | - Jack D Griffith
- Lineberger Comprehensive Cancer Center and Departments of Microbiology and Immunology, and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, United States
| | - Dirk Remus
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, United States
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40
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Xia Y, Fujisawa R, Deegan TD, Sonneville R, Labib KPM. TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans. EMBO J 2021; 40:e108053. [PMID: 34269473 PMCID: PMC8408604 DOI: 10.15252/embj.2021108053] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Revised: 06/10/2021] [Accepted: 06/11/2021] [Indexed: 12/30/2022] Open
Abstract
The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS-TIPIN, CUL-2LRR-1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM-7 subunit of CMG. Subsequently, the UBXN-3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4. We show that UBXN-3 is important in vivo for replisome disassembly in the absence of TIMELESS-TIPIN. Correspondingly, co-depletion of UBXN-3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN-3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.
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Affiliation(s)
- Yisui Xia
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Ryo Fujisawa
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Tom D Deegan
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Remi Sonneville
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Karim P M Labib
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
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41
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Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1. Int J Mol Sci 2021; 22:ijms22168796. [PMID: 34445496 PMCID: PMC8395760 DOI: 10.3390/ijms22168796] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 08/11/2021] [Accepted: 08/12/2021] [Indexed: 12/30/2022] Open
Abstract
Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.
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42
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Deol KK, Strieter ER. The ubiquitin proteoform problem. Curr Opin Chem Biol 2021; 63:95-104. [PMID: 33813043 PMCID: PMC8384647 DOI: 10.1016/j.cbpa.2021.02.015] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 02/22/2021] [Accepted: 02/25/2021] [Indexed: 12/23/2022]
Abstract
The diversity of ubiquitin modifications is immense. A protein can be monoubiquitylated, multi-monoubiquitylated, and polyubiquitylated with chains varying in size and shape. Ubiquitin itself can be adorned with other ubiquitin-like proteins and smaller functional groups. Considering different combinations of post-translational modifications can give rise to distinct biological outcomes, characterizing ubiquitylated proteoforms of a given protein is paramount. In this Opinion, we review recent advances in detecting and quantifying various ubiquitin proteoforms using mass spectrometry.
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Affiliation(s)
- Kirandeep K Deol
- Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA
| | - Eric R Strieter
- Department of Chemistry, University of Massachusetts, Amherst, MA, 01003, USA; Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, 01003, USA
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43
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Le TT, Ainsworth J, Polo Rivera C, Macartney T, Labib KP. Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes. Biochem J 2021; 478:2825-2842. [PMID: 34195792 PMCID: PMC8331092 DOI: 10.1042/bcj20210315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 06/25/2021] [Accepted: 06/29/2021] [Indexed: 11/17/2022]
Abstract
Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.
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Affiliation(s)
- Thanh Thi Le
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Johanna Ainsworth
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Cristian Polo Rivera
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Thomas Macartney
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Karim P.M. Labib
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
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44
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Xia Y. The Fate of Two Unstoppable Trains After Arriving Destination: Replisome Disassembly During DNA Replication Termination. Front Cell Dev Biol 2021; 9:658003. [PMID: 34368118 PMCID: PMC8335557 DOI: 10.3389/fcell.2021.658003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2021] [Accepted: 06/14/2021] [Indexed: 12/30/2022] Open
Abstract
In eukaryotes, the perfect duplication of the chromosomes is executed by a dynamic molecular machine called the replisome. As a key step to finishing DNA replication, replisome disassembly is triggered by ubiquitylation of the MCM7 subunit of the helicase complex CMG. Afterwards, the CDC48/p97 "unfoldase" is recruited to the ubiquitylated helicase to unfold MCM7 and disassemble the replisome. Here we summarise recently discovered mechanisms of replisome disassembly that are likely to be broadly conserved in eukaryotes. We also discuss two crucial questions that remain to be explored further in the future. Firstly, how is CMG ubiquitylation repressed by the replication fork throughout elongation? Secondly, what is the biological significance of replisome disassembly and what are the consequences of failing to ubiquitylate and disassemble the CMG helicase?
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Affiliation(s)
- Yisui Xia
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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45
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Basu S, Mak T, Ulferts R, Wu M, Deegan T, Fujisawa R, Tan KW, Lim CT, Basier C, Canal B, Curran JF, Drury LS, McClure AW, Roberts EL, Weissmann F, Zeisner TU, Beale R, Cowling VH, Howell M, Labib K, Diffley JFX. Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase. Biochem J 2021; 478:2481-2497. [PMID: 34198328 PMCID: PMC8286817 DOI: 10.1042/bcj20210219] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 05/05/2021] [Accepted: 05/10/2021] [Indexed: 12/15/2022]
Abstract
The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.
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Affiliation(s)
- Souradeep Basu
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Tiffany Mak
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Rachel Ulferts
- Cell Biology of Infection Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Mary Wu
- High Throughput Screening, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Tom Deegan
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Ryo Fujisawa
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Kang Wei Tan
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Chew Theng Lim
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Clovis Basier
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Berta Canal
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Joseph F Curran
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Lucy S Drury
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Allison W McClure
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Emma L Roberts
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Florian Weissmann
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Theresa U Zeisner
- Cell Cycle Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Rupert Beale
- Cell Biology of Infection Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Victoria H Cowling
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - Michael Howell
- High Throughput Screening, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
| | - Karim Labib
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K
| | - John F X Diffley
- Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, U.K
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46
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Fan Y, Köberlin MS, Ratnayeke N, Liu C, Deshpande M, Gerhardt J, Meyer T. LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components. J Cell Biol 2021; 220:212186. [PMID: 34037657 PMCID: PMC8160578 DOI: 10.1083/jcb.202009147] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 03/30/2021] [Accepted: 05/04/2021] [Indexed: 11/22/2022] Open
Abstract
After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45–MCM–GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.
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Affiliation(s)
- Yilin Fan
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA.,Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY
| | - Marielle S Köberlin
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA
| | - Nalin Ratnayeke
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA.,Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY
| | - Chad Liu
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA
| | - Madhura Deshpande
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY
| | - Jeannine Gerhardt
- Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY.,Department of Obstetrics and Gynecology, Weill Cornell Medicine, New York, NY
| | - Tobias Meyer
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA.,Department of Cell and Developmental Biology, Weill Cornell Medicine, New York, NY
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47
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Spotlight on the Replisome: Aetiology of DNA Replication-Associated Genetic Diseases. Trends Genet 2021; 37:317-336. [DOI: 10.1016/j.tig.2020.09.008] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 09/07/2020] [Accepted: 09/09/2020] [Indexed: 12/26/2022]
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48
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Abstract
The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.
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Affiliation(s)
- Ilan Attali
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA;
| | - Michael R Botchan
- Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
| | - James M Berger
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA;
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49
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Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie‐A‐Ling M, Lacaud G, Labib KPM. CUL2 LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle. EMBO Rep 2021; 22:e52164. [PMID: 33590678 PMCID: PMC7926238 DOI: 10.15252/embr.202052164] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 12/16/2020] [Accepted: 01/07/2021] [Indexed: 11/26/2022] Open
Abstract
The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
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Affiliation(s)
- Fabrizio Villa
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Ryo Fujisawa
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Johanna Ainsworth
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Kohei Nishimura
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
- Division of Biological ScienceGraduate School of ScienceNagoya UniversityNagoyaJapan
| | - Michael Lie‐A‐Ling
- Cancer Research U.K. Manchester InstituteThe University of ManchesterAlderley ParkUK
| | - Georges Lacaud
- Cancer Research U.K. Manchester InstituteThe University of ManchesterAlderley ParkUK
| | - Karim PM Labib
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
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50
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Mirsanaye AS, Typas D, Mailand N. Ubiquitylation at Stressed Replication Forks: Mechanisms and Functions. Trends Cell Biol 2021; 31:584-597. [PMID: 33612353 DOI: 10.1016/j.tcb.2021.01.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 01/18/2021] [Accepted: 01/21/2021] [Indexed: 11/17/2022]
Abstract
Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome.
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Affiliation(s)
- Ann Schirin Mirsanaye
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark
| | - Dimitris Typas
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark
| | - Niels Mailand
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark; Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark.
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