Ubiquitylation pathways controlling the end of chromosome replication
Lead Research Organisation:
University of Dundee
Department Name: UNLISTED
Abstract
Our cells contain a molecular machine, known as the ‘replisome’, which makes a precise copy of each of chromosomes before the cell divides in two, so that both of the ‘daughter cells’ contain all of the genetic information that is needed for life. The replisome is very carefully controlled, so that cells only make one single copy of the genetic blueprint that is contained in the chromosomes. If this regulation goes wrong, the results can be lethal in the most extreme case, or else can promote genetic diseases such as cancer.
There is much still to learn about the regulation of the replisome. We study how cells destroy the replisome after it has completed its job, by marking the replisome for destruction by a process known as ‘ubiquitylation’. We aim to identify the molecular machines that ubiquitylate the replisome and thus drive its destruction, and we also want to know how this dangerous process is controlled, so that it only happens at the right time. This represents a fundamental issue in cell biology, and is also likely to shed new light on our understanding of cancer development, and potentially might suggest new ideas for future cancer treatments.
There is much still to learn about the regulation of the replisome. We study how cells destroy the replisome after it has completed its job, by marking the replisome for destruction by a process known as ‘ubiquitylation’. We aim to identify the molecular machines that ubiquitylate the replisome and thus drive its destruction, and we also want to know how this dangerous process is controlled, so that it only happens at the right time. This represents a fundamental issue in cell biology, and is also likely to shed new light on our understanding of cancer development, and potentially might suggest new ideas for future cancer treatments.
Technical Summary
My group studies the mechanisms and regulation of the eukaryotic replisome – the molecular machine that produces a single copy of the chromosomes in each cell cycle. Replisome biology is defective in many human tumours, providing opportunities for the development of new therapies. We discovered that disassembly of the eukaryotic replisome during DNA replication termination is a regulated process that is controlled by ubiquitylation of the DNA helicase known as CMG (Cdc45-MCM-GINS), around which the relisome is built. Ubiquitylation leads to disassembly of the CMG helicase, and thus of the replisome, dependent upon the Cdc48 ATPase that probably denatures the ubiquitylated subunit of CMG. Moreover, we found that metazoa have a second mechanism for replisome disassembly that acts during mitosis and is regulated by a factor mutated in a variety of human cancers, suggesting a potential future strategy for disease treatment. Our preliminary data indicates that there are yet more replisome disassembly pathways to define and characterize. We aim to study their mechanisms and regulation, and then investigate their links to human disease.
Organisations
- University of Dundee (Lead Research Organisation)
- Francis Crick Institute (Collaboration)
- University of Copenhagen (Collaboration)
- University of Manchester (Collaboration)
- HARVARD UNIVERSITY (Collaboration)
- Friedrich Miescher Institute for Biomedical Research (FMI) (Collaboration)
- Centre for Genomic Regulation (CRG) (Collaboration)
- National Institute of Genetics (Collaboration)
- Medical Research Council (MRC) (Collaboration)
- Columbia University (Collaboration)
- UNIVERSITY OF DUNDEE (Collaboration)
- MRC Harwell Institute (Collaboration)
People |
ORCID iD |
| Karim Labib (Principal Investigator) |
Publications
Basu S
(2021)
Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase.
in The Biochemical journal
Canal B
(2021)
Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.
in The Biochemical journal
Canal B
(2021)
Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.
in The Biochemical journal
Deegan TD
(2019)
Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.
in Molecular cell
Deng L
(2019)
Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.
in Molecular cell
Evrin C
(2022)
Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II.
in The EMBO journal
Evrin C
(2018)
Histone H2A-H2B binding by Pol a in the eukaryotic replisome contributes to the maintenance of repressive chromatin.
in The EMBO journal
Evrin C
(2023)
DONSON is required for CMG helicase assembly in the mammalian cell cycle.
in EMBO reports
Related Projects
| Project Reference | Relationship | Related To | Start | End | Award Value |
|---|---|---|---|---|---|
| MC_UU_00018/1 | 31/03/2018 | 31/03/2024 | £4,394,000 | ||
| MC_UU_00018/2 | Transfer | MC_UU_00018/1 | 31/03/2018 | 31/03/2024 | £2,542,000 |
| MC_UU_00018/3 | Transfer | MC_UU_00018/2 | 31/03/2018 | 31/03/2024 | £3,121,000 |
| MC_UU_00018/4 | Transfer | MC_UU_00018/3 | 31/03/2018 | 31/03/2024 | £2,751,000 |
| MC_UU_00018/5 | Transfer | MC_UU_00018/4 | 31/03/2018 | 31/03/2024 | £3,744,000 |
| MC_UU_00018/6 | Transfer | MC_UU_00018/5 | 31/03/2018 | 31/03/2024 | £2,520,000 |
| MC_UU_00018/7 | Transfer | MC_UU_00018/6 | 31/03/2018 | 31/03/2024 | £2,557,000 |
| MC_UU_00018/8 | Transfer | MC_UU_00018/7 | 31/03/2018 | 31/03/2024 | £2,128,000 |
| Description | Chair of EMBO Installation Grant Committee |
| Geographic Reach | Europe |
| Policy Influence Type | Participation in a guidance/advisory committee |
| Impact | This scheme helps member countries to recruit and retain some of the best young scientists in the world and also helps them to attract international funding. |
| URL | https://www.embo.org/funding/fellowships-grants-and-career-support/installation-grants/ |
| Description | Chair of EMBO Lab Sustainability Award Advisory Board |
| Geographic Reach | Europe |
| Policy Influence Type | Participation in a guidance/advisory committee |
| URL | https://www.embo.org/the-embo-communities/embo-lab-sustainability-award/ |
| Description | Head of Climate Action and Sustainability for School of Life Sciences, University of Dundee |
| Geographic Reach | Local/Municipal/Regional |
| Policy Influence Type | Contribution to new or improved professional practice |
| Impact | See contributions described above. |
| Description | Member of Wellcome Trust's Sir Henry Dale Fellowship Selection Committee |
| Geographic Reach | Europe |
| Policy Influence Type | Membership of a guideline committee |
| Description | Panel member for ERC funding panel LS1 - Molecules of Life |
| Geographic Reach | Europe |
| Policy Influence Type | Membership of a guideline committee |
| Description | CRUK Programme Grant |
| Amount | £1,994,243 (GBP) |
| Funding ID | C578/A24558 |
| Organisation | Cancer Research UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2018 |
| End | 08/2023 |
| Description | MRC Climate Crisis Network |
| Amount | £198,000 (GBP) |
| Funding ID | 119585 |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 01/2022 |
| End | 12/2023 |
| Description | Mechanism of metazoan replisome assembly in health and disease |
| Amount | £3,250,184 (GBP) |
| Funding ID | 302391/Z/23/Z |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2024 |
| End | 03/2032 |
| Description | Regulation of metazoan replisome disassembly at stalled replication forks and during mitosis |
| Amount | £188,296,973 (GBP) |
| Funding ID | DRCRPG-Nov22/100016 |
| Organisation | Cancer Research UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2023 |
| End | 08/2028 |
| Description | Sir Henry Wellcome postdoctoral fellowship (awarded to Dr. Tom Deegan in my group) |
| Amount | £250,000 (GBP) |
| Organisation | Wellcome Trust |
| Department | Wellcome Trust Bloomsbury Centre |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 04/2017 |
| End | 04/2022 |
| Description | Understanding the mechanism of mitotic replisome disassembly |
| Amount | ￥13,456,000 (JPY) |
| Organisation | Japan Society for the Promotion of Science (JSPS) |
| Sector | Public |
| Country | Japan |
| Start | 03/2022 |
| End | 02/2024 |
| Description | WT Senior Investigator Award |
| Amount | £1,849,504 (GBP) |
| Funding ID | 102943/Z/13/Z |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2014 |
| End | 03/2019 |
| Title | C. elegans strains to study the regulation of chromosome replication |
| Description | Many C. elegans strains were generated in the course of our project recently published in The EMBO Journal. |
| Type Of Material | Model of mechanisms or symptoms - non-mammalian in vivo |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Xia Y, Fujisawa R, Deegan TD, Sonneville R, Labib K. (2021) TIMELESS - TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans. EMBO J., 40(17):e108053. doi: 10.15252/embj.2021108053 |
| URL | https://www.embopress.org/doi/full/10.15252/embj.2021108053 |
| Title | Mammalian cell lines with which to purify the replisome |
| Description | Cells with tagged replisome components allow the purification of the replisome. |
| Type Of Material | Biological samples |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Publications. |
| URL | https://mrcppureagents.dundee.ac.uk/ |
| Title | Plasmids to express replisome components or modulate their expression via CRISPR or RNAi |
| Description | A range of plasmids have been generated that either allow expression in budding yeast of recombinant components of the metazoan replisome, or that allow targeting of replisome loci via CRISPR, or that allow the level of replisome proteins to be modulated by RNAi. |
| Type Of Material | Biological samples |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Valuable tools have been made available to the research community. |
| URL | https://mrcppureagents.dundee.ac.uk/ |
| Title | Sheep polyclonal antibodies to protein components of the metazoan replisome (C. elegans / mouse / human) |
| Description | More than 30 sheep polyclonal antibodies have been generated to various components of the metazoan replisome. |
| Type Of Material | Antibody |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | Valuable tools have been made available to the research community via the MRC PPU reagents and services website. |
| URL | https://mrcppureagents.dundee.ac.uk/ |
| Description | Analysis of chromatin replication in C. elegans |
| Organisation | Centre for Genomic Regulation (CRG) |
| Country | Spain |
| Sector | Academic/University |
| PI Contribution | We have used CRISPR to generate worms with mutations in histone-binding motifs of the eukaryotic replisome. |
| Collaborator Contribution | Ben Lehner's group will analyse our mutant worms to screen for defects in the maintenance of repressive chromatin |
| Impact | None yet |
| Start Year | 2017 |
| Description | Collaborative screen for small molecule inhibitors of SARS-CoV-2 |
| Organisation | Francis Crick Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We expressed and purified three enzymes of SARS-CoV-2 and established a high throughput assay to monitor activity of the Nsp15 nuclease. |
| Collaborator Contribution | Collaboration with groups of John Diffley and Paul Nurse at the Francis Crick institute, to establish assays to monitor activity of all enzyme components of SARS-CoV-2 and then screen a library of small molecules for inhibitors. |
| Impact | Canal B, Fujisawa R, McClure AW, Deegan TD, Wu M, Ulferts R, Weissmann F, Drury LS, Bertolin AP, Zeng J, Beale R, Howell M, Labib K, Diffley JFX. (2021) Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease. Biochem J., 478, 2465-2479 Lim CT, Tan KW, Wu M, Ulferts R, Armstrong LA, Ozono E, Drury LS, Milligan JC, Zeisner TU, Zeng J, Weissmann F, Canal B, Bineva-Todd G, Howell M, O'Reilly N, Beale R, Kulathu Y, Labib K, Diffley JFX. (2021) Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease. Biochem J., 478, 2517-2531. Canal B, McClure AW, Curran JF, Wu M, Ulferts R, Weissmann F, Zeng J, Bertolin AP, Milligan JC, Basu S, Drury LS, Deegan TD, Fujisawa R, Roberts EL, Basier C, Labib K, Beale R, Howell M, Diffley JFX. (2021) Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease. Biochem J., 478, 2445-2464. Milligan JC, Zeisner TU, Papageorgiou G, Joshi D, Soudy C, Ulferts R, Wu M, Lim CT, Tan KW, Weissmann F, Canal B, Fujisawa R, Deegan T, Nagaraj H, Bineva-Todd G, Basier C, Curran JF, Howell M, Beale R, Labib K, O'Reilly N, Diffley JFX. (2021) Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease. Biochem J 478, 2499-2515. 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. (2021) Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase. Biochem J 478, 2481-2497. |
| Start Year | 2020 |
| Description | Growth and genetic modification of mouse embryonic stem cells |
| Organisation | University of Manchester |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Until recently, our studies of chromosome replication were all limited to the budding yeast S. cerevisiae. We are now extending our work into higher eukaryotic models, including mouse embryonic stem cells. As part of this, we have collaborated with Dr. Georges Lacaud at the University of Manchester, who is a renowned expert in the growth, genetic modification, and in vitro differentiation of mouse ES cells. |
| Collaborator Contribution | Dr. Lacaud's lab have provided expertise in the growth, genetic modification, and in vitro differentiation of mouse ES cells. |
| Impact | Villa, F., Fujisawa, R., Ainsworth, J., Nishimura, K., Lie-A-Ling, M., Lacaud, G. & Labib, K. (2021) CUL2 LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle. EMBO Rep. e52164. doi: 10.15252/embr.202052164. Online ahead of print |
| Start Year | 2015 |
| Description | Histone occupancy across the yeast genome after mutation of Spt5 |
| Organisation | Columbia University |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | The group of Dr. Zhang at Columbia are world leaders in monitoring histone occupancy across the genome during chromosome replication and transcription. We prevoiusly collaborated to study the role of the Mcm2 helicase subunit in redepositing parental histones onto nascent DNA (Gan et al, 2018). In the present collaboration we have studied the phenotype of mutating a novel histone-binding activity that we have identified in the RNA polymerase II transcriptional machinery. |
| Collaborator Contribution | ChIP-Seq analysis and related approaches, to study histone occupancy across the yeast genome, in mutant strains that are generated in my group (mutation of a novel histone-binding motif in Spt5). |
| Impact | Gan, H., Serra-Cardona, A., Hua, X, Zhou, H., Labib, K., Yu, C. and Zhang, Z. (2018) The Mcm2-Ctf4-Pola axis facilitates parental histone H3-H4 transfer to lagging strands. Mol. Cell, 72, 140-151. Evrin, C., Serra-Cardona, A., Duan, S., Mukherjee, P.P., Zhang, Z. and Labib, K.P.M. (2022) Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II. EMBO J., e109783. Doi: 10.15252/embj.2021109783. |
| Start Year | 2018 |
| Description | Induced protein degradation via auxin degron technology |
| Organisation | National Institute of Genetics |
| Country | Japan |
| Sector | Academic/University |
| PI Contribution | We will generate auxin degron alleles in yeast and mouse ES cells, using agents provided by the group of Professor Masato Kanemaki at NIG. |
| Collaborator Contribution | The group of Professor Masato Kanemaki at NIG will provide the reagents that are needed for the generation of auxin degrons in yeast and mouse ES cells. |
| Impact | Evrin et al, EMBO J., 2022 |
| Start Year | 2021 |
| Description | MRC Harwell |
| Organisation | MRC Harwell |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Engage in discussion about mouse lines |
| Collaborator Contribution | Supply of mouse lines to investigate PD mutations and the Rab pathway |
| Impact | On going |
| Start Year | 2015 |
| Description | Processing of incomplete DNA replication during mitosis in human cells |
| Organisation | University of Copenhagen |
| Country | Denmark |
| Sector | Academic/University |
| PI Contribution | We discovered that the TRAIP E3 ligase is required to process incomplete DNA replication in C. elegans embryos. |
| Collaborator Contribution | Based on our findings, our partners tested whether the TRAIP E3 ligase is required to process incomplete DNA replication in human cells. |
| Impact | Sonneville R, Bhowmick R, Hoffmann S, Mailand N, Hickson ID, Labib K. (2019). TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.. eLife |
| Start Year | 2018 |
| Description | Structural biology of E3 ligases in association with the replisome |
| Organisation | Friedrich Miescher Institute for Biomedical Research (FMI) |
| Country | Switzerland |
| Sector | Academic/University |
| PI Contribution | We are defining protein complexes that Nico Thomma's group at FMI in Basel will study by cryo-EM. |
| Collaborator Contribution | Nico Thomma's group at FMI in Basel will produce proteins and study structures by cryo-EM. |
| Impact | None yet |
| Start Year | 2018 |
| Description | Structural biology of the replisome |
| Organisation | Medical Research Council (MRC) |
| Department | MRC Laboratory of Molecular Biology (LMB) |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have defined protein complexes for analysis by cryo-EM. |
| Collaborator Contribution | Joe Yeeles group at the MRC LMB in Cambridge puts our protein complexes on cryoEM grids and then aims to solve structures. |
| Impact | Jenkyn-Bedford et al, Nature, 2021 Xia et al, Science, 2023 |
| Start Year | 2019 |
| Description | Studying chromosome replication dynamics by DNA fibre analysis and High Content Microscopy |
| Organisation | University of Dundee |
| Department | College of Life Sciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We are studying novel DNA replication factors in animal cells. |
| Collaborator Contribution | Dr. Constance Alabert is a world leader in the analysis of DNA replication dynamics by DNA fibre analysis ('DNA combing') and 'high content microscopy' (also known as Quantitative Imaging Based Cytometry). |
| Impact | Xia et al, Science, 2023 Evrin et al, EMBO Rep., 2023 |
| Start Year | 2022 |
| Description | TRAIP and mitotic CMG helicase disassembly |
| Organisation | Harvard University |
| Department | Harvard Medical School |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | We showed the worm orthologue of the mammalian TRAIP E3 ligase is required for CMG helicase disassembly during mitosis. |
| Collaborator Contribution | They showed the Xenopus orthologue of the mammalian TRAIP E3 ligase is required for CMG helicase disassembly during mitosis. |
| Impact | N.A. |
| Start Year | 2018 |
| Description | A school talk (Broughton High School, Edinburgh) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | School talk ('The Origins of Life') |
| Year(s) Of Engagement Activity | 2023 |
| URL | https://rse.org.uk/whats-on/schools/ |
| Description | School Visit (Calderside Academy, Glasgow) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | Talk about 'Origins of Life on Earth' to S5 and S6 higher biology students. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://rse.org.uk/whats-on/schools/ |
| Description | School Visit (Jordanhill school, Glasgow) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | Talk about "Origins of Life on Earth' to S3 and S4 biology students. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://rse.org.uk/whats-on/schools/ |
| Description | School Visit (Kirkwall, Orkney) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Talk about "Origins of Life on Earth' given to two higher biology classes. |
| Year(s) Of Engagement Activity | 2018 |
| Description | School Visit (Kirkwall, Orkney) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | I took my entire research group to meet the higher biology students at Kirkwall Grammar School, Kirkwall, Orkney, to speak about "How and why to be a scientist?" |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk to Public (The Origins of Life on Earth) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | Invited talk at the 'Orkney International Science Festival' 2018. |
| Year(s) Of Engagement Activity | 2018 |
| URL | http://oisf.org/programme-2018/ |