Chromosome Replication and the Regulation of Genome Integrity

Lead Research Organisation: University of Dundee

Abstract

Life begins from a single cell, which has the ability to reproduce itself many times over, allowing the formation of complex creatures such as ourselves. To survive cutting itself in two, a cell must first make an almost perfect copy of the chromosomes that contain all the information that is needed to build each living creature, so that each of the two ‘daughter cells’ can receive an intact set of chromosomes. Defects in the process of chromosome duplication drive the early stages of cancer development, and make cancer cells sensitive to therapies that interfere with chromosome replication. We are studying the mechanisms and regulation of chromosome duplication, which is one of the most complicated processes in cell biology, so that many important questions remain to be answered. A better understanding of this process in normal cells will make it possible to investigate how chromosome replication becomes defective in cancer cells. Ultimately, this will help to guide the design of future and more effective anti-cancer therapies.

Technical Summary

My group aims to understand how the molecular machinery at DNA replication forks allows eukaryotic cells to preserve their highly complex chromosomes from one generation to the next. One of the most important challenges for proliferating cells is to make a single and precise copy of each chromosome before cell division. In eukaryotes this is a particularly complex task, involving the duplication of vast amounts of DNA, the reproduction of epigenetic chromatin modifications along each chromosome, and other tasks such as the establishment of cohesion between the two sister chromatids that are the products of replication. For these reasons, chromosome replication is one of the most complex and fascinating of all cellular processes in eukaryotes, with mechanisms and regulation that are still understood poorly in all species. A better understanding of chromosome replication is particularly important from the viewpoint of human cancer, since defects in chromosome replication are a very early feature in cancer development, providing a target for the future development of new therapies that might selectively kill cancer cells. Many of the proteins involved in chromosome replication associate with each other to form a multi-protein assembly known as the replisome, which physically connects the essential DNA helicase that unwinds the parental duplex, to the DNA polymerases and other factors. My group is focussed on defining the nature of eukaryotic replisome, understanding its mechanisms of action, and studying its regulation by post-translational modifications. Most of the proteins that we study have a single orthologue from humans to yeast, reflecting the very high degree of conservation of the eukaryotic replisome, and indicating the value of using simple experimental systems. Our work exploits the unique advantages of budding yeast for studies of chromosome replication, combining state of the art biochemical and genetic techniques, but we are also extending our work into other species where appropriate, including mammalian cells.

Publications

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Wu Y (2017) Targeting the Genome-Stability Hub Ctf4 by Stapled-Peptide Design. in Angewandte Chemie (International ed. in English)

 
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 Europe 
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 Quinquennial review panel for MRC Cancer Unit, University of Cambridge
Geographic Reach National 
Policy Influence Type Participation in a advisory 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 Wellcome Trust interview panel member
Geographic Reach National 
Policy Influence Type Participation in a advisory 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 03/2023
 
Description JSPS fellowship
Amount ¥10,500,000 (JPY)
Funding ID Award to Dr. Kohei Nishimura to work in my group 
Organisation Japan Society for the Promotion of Science (JSPS) 
Sector Public
Country Japan
Start 04/2014 
End 03/2016
 
Description Naito Foundation Research expenses award
Amount ¥3,000,000 (JPY)
Funding ID Award to Dr. Kohei Nishimura to work in my group 
Organisation Naito Foundation 
Sector Charity/Non Profit
Country Japan
Start 04/2014 
End 03/2016
 
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 A new family of deubiquitylating enzymes. 
Organisation University of Dundee
Department MRC Protein Phosphorylation and Ubiquitylation Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution We have collaborated with the group of Yogesh Kulathu to characterise a new family of deubiquitylating enzymes. Pedro Nkosi in my group has explored the role of this new deubiquitylase in budding yeast.
Collaborator Contribution Yogesh Kulathu discovered this new family of deubiquitylating enzymes and his group has made the major contribution in characterising its structure and function.
Impact A manuscript has just been submitted for publication.
Start Year 2015
 
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 Global analysis of histone occupancy across the yeast genome 
Organisation Fred Hutchinson Cancer Research Center (FHCRC)
Country United States 
Sector Charity/Non Profit 
PI Contribution We are studying how the chromosome replication machinery influences the chromatin landscape and its regeneration during replication. In 2016 we initiated a collaboration with the group of Toshio Tsukiyama, a world leader in the global analysis of histone occupancy across the yeast genome, and its regulation by the transcriptional machinery. Dr. Tsukiyama's group has used ChIP-Seq of histone H3 to monitor histone occupancy in mutants generated in my group.
Collaborator Contribution Toshio's lab has performed ChIP-Seq of histone H3 to monitor histone occupancy in mutants generated in my group.
Impact The data were useful and contributed to an ongoing project in my group, which we hope to write up in 2019.
Start Year 2016
 
Description How the chromosome replication machinery deals with topological issues during the final stages of DNA synthesis. 
Organisation University of Sussex
Country United Kingdom 
Sector Academic/University 
PI Contribution We are studying how the chromosome replication machinery deals with topological issues during the final stages of DNA synthesis. Dr. Baxter is an expert in the analysis of DNA intermediates during replication termination. We collaborate with Dr. Baxter's group at the University of Sussex, to study DNA replication termination both in vivo (Dr. Baxter's group and Dr. Maria Angeles Ortiz Bazan in my group) and in vitro (Dr. Tom Deegan in my group).
Collaborator Contribution Dr. Baxter uses 1D and 2D agarose gels to study the termination of plasmid DNA replication in vivo, in a range of mutant strains generated in my lab by Dr. Ortiz Bazan.
Impact Deegan TD, Baxter J, Ortiz Bazán MÁ, Yeeles JTP, Labib KPM. (2019). Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.. Molecular cell, 74 (2), pp. 231-244.e9
Start Year 2015
 
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 Mapping ubiquitylation sites by mass spectrometry 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution We have purified a ubiquitylated subunit of the DNA helicase that unwinds replication forks, and need to map the sites by mass spectrometry. We are collaborating with the group of Ron Hay to do this, as they are world leaders in this area.
Collaborator Contribution Ron Hay's group have mapped sites of ubiquitylation in replication factors for us by mass spectrometry.
Impact A manuscript regarding this collaborative work will be submitted within a month.
Start Year 2014
 
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 Role of leading strand DNA polymerase in checkpoint signalling pathway 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We collaborated with the group of Dr. Giacomo de Piccoli, to study the regulation of DNA polymerase epsilon by the S-phase checkpoint pathway. We found that the association of DNA polymerase epsilon with a factor called Ctf18-RFC is necessary for activation of the S-phase checkpoint, in response to defects in chromosome replication.
Collaborator Contribution Dr. de Piccoli's group helped us with experiments that were needed to address comments raised by reviewers of our manuscript.
Impact We published a paper concerning this issue in 2015 (Garcia-Rodriguez et al, N.A.R., 2015).
Start Year 2014
 
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 Studying chromatin regeneration during DNA replication in higher eukaryotes 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Academic/University 
PI Contribution We are studying how the chromosome replication machinery helps to regenerate chromatin during the process of DNA replication. Initially our work has all been in budding yeast, but we are now starting to extend these studies into higher eukaryotic models. As part of this work, we will collaborate with Dr. Simon Boulton at the Crick institute. My group will characterise mutations in replication factors, which Simon's group will then mutate in mice.
Collaborator Contribution Dr. Simon Boulton's group will characterise mutations in mice, corresponding to mutations in DNA replication factors that my group have defined biochemically.
Impact None yet.
Start Year 2016
 
Description Studying chromosome replication in worm early embryos 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution We use the early embryo of the worm C. elegans as a model system for studying the metazoan replisome. Dr. Anton Gartner at the University of Dundee is a renowned expert in studies of genome stability and cell cycle in C. elegans, and so we have collaborated on projects where our interests have overlapped (e.g. mechanisms that help cells complete genome duplication and segregation before cell division).
Collaborator Contribution Dr. Gartner's group provide invaluable advice and reagents for our work.
Impact Sonneville, R., Craig, G., Labib, K., Gartner, A. and Blow, J. J. (2015). Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos. Cell Rep. 12, 405-417. Hong Y, Velkova M, Silva N, Jagut M, Scheidt V, Labib K, Jantsch V, Gartner A. (2018) The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans. PLoS Genet. 14(6):e1007453. doi: 10.1371/journal.pgen.1007453. Hong, Y., Sonneville, R., Wang, B., Scheidt, V., Meier, B., Woglar, A., Demetriou, S., Labib, K., Jantsch, V., and Gartner, A. (2018) LEM-3 is a midbody-tethered nuclease that resolves chromatin bridges during late mitosis. Nat. Commun. 9, 728. doi: 10.1038/s41467-018-03135-w.
Start Year 2014
 
Description Tethering of the Mms22-Rtt101 ligase to the replisome by Ctf4 
Organisation Johannes Gutenberg University of Mainz
Department Institute of Molecular Biology (IMB), Mainz, Germany
Country Germany 
Sector Academic/University 
PI Contribution We collaborate with the group's of Matthias Peter and Brian Luke, to study the regulation of genome integrity by ubiquitylation. We helped them to show that the E3 ligase Mms22-Rtt101 is tethered to the replisome by the Ctf4 protein.
Collaborator Contribution The groups of Matthias Peter and Brian Luke have studied the role of the Mms22-Rtt101 ligase at defective DNA replication forks.
Impact Buser, R., Kellner, V., Melnik, A., Wilson-Zbinden, C., Schellhaas, R., Kastner, L., Piwko, W., Dees, M., Picotti, P., Maric, M., Labib, K., Luke, B. and Peter, M. (2016) The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress. PLOS Genetics, DOI:10.1371/journal.pgen.1005843.
Start Year 2013
 
Description The role of the Cul2-LRR1 E1 ligase at the end of chromosome replication in Xenopus laevis 
Organisation University of Birmingham
Department Institute of Cancer and Genomic Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We showed that the Cul2-LRR1 E1 ligase controls the final stages of chromosome replication in the early embryo of the worm C. elegans. Our work suggests that this ligase might play an equivalent role in all higher eukaryotes including humans. Frog eggs provide the best experimental system to test this idea, as anything conserved from worm to frog is almost certainly conserved in humans too (but frog eggs provide a much better experimental system to study chromosome replication than human cells.). We have thus generated reagents and initiated a collaboration with the group of Dr. Aga Gambus in Birmingham, whose group specialises in the study of chromosome replication in frog egg extracts.
Collaborator Contribution The group of Dr. Aga Gambus in Birmingham specialises in the study of chromosome replication in frog egg extracts. They will perform key experiments with reagents and ideas generated here in Dundee.
Impact https://www.nature.com/articles/ncb3500
Start Year 2015
 
Description Discovery Day (talk to general public in Dundee) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk sparked questions and discussion afterwards.

Video of the talk was prepared for release on 'YouTube'
Year(s) Of Engagement Activity 2014
URL http://www.dundee.ac.uk/revealingresearch/newsandevents/dd14/
 
Description MRC PPU website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I completely re-wrote and re-designed the MRC PPU website, in advance of our quinquennial review.
Year(s) Of Engagement Activity 2017
URL https://www.ppu.mrc.ac.uk
 
Description MRC-PPU Collaboration with Baldragon Academy 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The Medical Research Council's Protein Phosphorylation Ubiquitylation Unit (MRCPPU), part of The University of Dundee, has prioritized public engagement in an effort to engage the general public and ensure that the research activities and breakthroughs are communicated to the community. Of equal importance in these communication efforts is educational outreach to students within the Dundee community.

Thus, during session 2014 -2015 and 2015-2016 school year, the MRC-PPU will partner with a local secondary school -- Baldragon Academy (BA). Teachers in BA's Science Department will collaborate with scientists at the MRC-PPU in an educational outreach effort (see Appendix 1). The purpose of this project is to increase interest and engagement in science and related careers. It will be starting in August 2014 with the S1 pupils.

Scientists from the unit will be working with the pupils on a monthly basis at the school during their science classes and will be providing them with opportunities to take part in various science experiments and demonstrations (aligned with Scotland's Curriculum for Excellence). The scientists are leaders in their field of research and as such come from all over the world. They are currently based in Dundee.

Thus far, student have been very enthusiastic about the labs and very receptive to the volunteers. They have asked a multitude of questions and have even asked volunteers back to visit. We have also had numerous students of different ages from the school ask to participate in work experience activities to learn more about the Unit and science in general.
Year(s) Of Engagement Activity 2014,2015
 
Description Media interest (DNA replication study) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release

Article in 'The Courier Mail' (Australia), and 'The Evening Telegraph".
Year(s) Of Engagement Activity 2014
URL http://www.eveningtelegraph.co.uk/news/local/dundee/cancer-fight-takes-another-step-forward-1.653042
 
Description Orkney International Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Talk sparked questions and discussion afterwards

I was invited to talk to biology students at the local high school (15-16 year olds).
Year(s) Of Engagement Activity 2014
URL http://oisf.org
 
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 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 to biology students ("A brief history of life"), Kirkwall Grammar School, Orkney.
Year(s) Of Engagement Activity 2017
 
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 sparked questions and discussion.

N/A
Year(s) Of Engagement Activity 2014
 
Description Seminar to general public 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Seminar to general public ("A brief history of life"), as part of the Orkney International Science Festival 2017, Kirkwall, Orkney.
Year(s) Of Engagement Activity 2017
URL http://oisf.org/programme-2017/
 
Description Seminar to general public 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Seminar to general public ("A brief history of life"), as part of the 'Café Science' series at the University of Dundee.
Year(s) Of Engagement Activity 2017
URL https://www.cafesciencedundee.co.uk
 
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/
 
Description YouTube video about our work and its links to cancer 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Recording of interview led to production of short 'YouTube' video, about our work and its links to cancer.

Production of short 'YouTube' video, about our work and its links to cancer.
Year(s) Of Engagement Activity 2014
URL https://www.youtube.com/watch?v=NQOyuZrfgSc
 
Description YouTube video of talk to public about our research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Talk sparked questions and discussion afterwards.

Video of talk released on YouTube.
Year(s) Of Engagement Activity 2014
URL https://www.youtube.com/watch?v=g1ycEnm9uR0