Ubiquitylation pathways controlling the end of chromosome replication

Lead Research Organisation: University of Dundee

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.

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.
 
Description Cell and Molecular Biology 'Sectional Committee' for selection of new fellows of the Royal Society of Edinburgh
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
URL https://www.royalsoced.org.uk
 
Description Chair of EMBO Young Investigator Programme Selection Committee
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
URL http://www.embo.org/about-embo/committees#yip
 
Description Member of Wellcome Trust's Sir Henry Dale Fellowship Selection Committee
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
 
Description Senior Advisory Board of 'CNRS Institute of Molecular Genetics' (IGMM), Montpellier, France.
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
URL http://www.igmm.cnrs.fr/?lang=en
 
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 03/2023
 
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 05/2017 
End 04/2022
 
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 04/2014 
End 03/2019
 
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 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 This collaboration has been fundamentally important to us, since it helped initiate our work with mouse ES cells. So far this work remains unpublished.
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 recently collaborated to study the role of the Mcm2 helicase subunit in redepositing parental histones onto nascent DNA (Gan et al, 2018). Now we are collaborating to study 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.
Start Year 2018
 
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 eukaryotic replisome 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborate with the group of Luca Pellegrini, in the Department of Biochemistry at the University of Cambridge, to study the structure and function of the replisome component Ctf4. My group has found that Ctf4 links the DNA helicase at replication forks to a list of other proteins.
Collaborator Contribution Dr. Pellegrini's group shares our interest in chromosome replication and takes structural approaches to studying the eukaryotic replisome (mostly via crystallography, but now also by cryo-EM), which complement our biochemical and biological studies of replisome function.
Impact Simon, A. C., Zhou, J. C., Perera, R. L., van Deursen, F., Evrin, C., Ivanova, M. E., Kilkenny, M. L., Renault, L., Kjaer, S., Matak-Vinkovic, D., Labib, K., Costa, A. and Pellegrini, L. (2014). A Ctf4 trimer couples the CMG helicase to DNA polymerase alpha in the eukaryotic replisome. Nature, 510, pp. 293-297. Villa, F., Simon, A. C., Ortiz-Bazan, M. A., Kilkenny, M. L., Wirthensohn, D., Wightman, M., Matak-Vinkovic, D., Pellegrini, L. and Labib, K. (2016) Ctf4 is a hub in the eukaryotic replisome that links multiple CIP-box proteins to the CMG helicase. Mol. Cell, 63, 1-12. Wu Y., Villa F., Maman J., Dobnikar L., Lau Y.H., Simon A.C., Labib K., Spring D.R. and Pellegrini L. (2017) Targeting the genome stability hub Ctf4 by stapled peptide design. Angew Chem Int Ed Engl. doi: 10.1002/anie.201705611.
Start Year 2012
 
Description Structural biology of the yeast replisome with associated E3 ligase 
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. My postdoc Tom Deegan is now pursuing this work in collaboration with the group of Joe Yeeles at LMB Cambridge.
Collaborator Contribution Joe Yeeles group has provided assistance and expertise to help Tom Deegan put protein complexes onto cryoEM grids. Joe's group has then imaged these grids at LMB, and also assisted Tom during a visit to the national facility at Diamond. Joe's lab are providing assistance with structural determination, using these data.
Impact None yet, but we expect a manuscript to be submitted over the coming year.
Start Year 2019
 
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 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 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 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/