Vertebrate mutagenesis and DNA damage tolerance

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

The lab is trying to understand how problems encountered as cells replicate their DNA lead to mutations and to epigenetic alterations which cause changes in the pattern of gene expression in cells, sometimes known as epimutations. Both mutations and epimutations are seen extensively in cancer cells and drive the development of the disease, so understanding their origin is central to understanding how cancer arises.
DNA can be damaged and can also fold into secondary structures, especially when it is unwound. Both can lead to interruption of DNA replication. Rescuing DNA synthesis in these circumstances requires the complex interaction of many proteins. Our primary aim is to understand how cells coordinate the replication of DNA impediments with maintaining the stability of their genomes and epigenomes.
Our studies make use of human, mouse and avian cell lines which we can easily modify genetically. A by-product of our studies has been the development of cell lines and techniques for the in vitro evolution of antibodies and other proteins to have specific binding characteristics, a technique that can be used to generate molecules of therapeutic utility.

Technical Summary

The group studies how problems encountered during DNA replication lead to both genetic mutation and to epimutation, changes in gene expression caused by local alterations in the chromatin of somatic cells.
DNA replication is readily interrupted by DNA damage and by naturally occurring secondary structures in the DNA. DNA damage frequently stalls the replicative DNA polymerases but can be replicated by specialised translesion DNA polymerases, which possess active sites that are capable of accommodating damaged or distorted DNA templates. While the use of these polymerases allows replication to be completed it also increases the chance of mutagenesis. A major goal of the lab has been to understand how and when these enzymes are deployed and how their activity is regulated to allow efficient and safe replication of damaged DNA.
We have shown that in addition to assisting in the replication of damaged DNA, some specialised polymerases, acting in concert with specialised DNA helicases, also play a role in the replication of DNA secondary structures. We have shown how replication stalling by DNA secondary structure formation (e.g. G quadruplex and triplex DNA) interrupts histone recycling and locally prevents the proper inheritance of histone modifications through cell division leading to altered gene expression. This observation has linked replication stress to disruption of epigenetic memory and to variations in gene expression, a feature of many cancer cells. We are working to further understand how DNA replication impediments impair epigenetically encoded transcriptional memory and the circumstances in which loss of coupling between DNA replication and the propagation of epigenetic information leads to disease.

Finally, our work, which predominantly uses somatic cell genetics, has also led to the identification and development of cell lines capable of continuous mutagenesis which we have used to evolve specific antibody specificities and other proteins of therapeutic interest.
 
Description AICR Project Grant
Amount £127,951 (GBP)
Organisation Association for International Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2008 
End 06/2011
 
Description AICR Project Grant
Amount £144,710 (GBP)
Funding ID 11-0514 
Organisation Association for International Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 12/2014
 
Description AstraZeneca/LMB Blue Skies Initiative
Amount £383,501 (GBP)
Funding ID BSF5 
Organisation AstraZeneca 
Department Research and Development AstraZeneca
Sector Private
Country United Kingdom
Start 05/2015 
End 02/2019
 
Description Cesar Milstein Studentship - Peris
Amount £110,000 (GBP)
Organisation British Society for the History of Science (BSHS) 
Department Darwin Trust of Edinburgh
Sector Academic/University
Country United Kingdom
Start 01/2015 
End 12/2018
 
Description Cesar Milstein Studentship - Romanello
Amount £96,000 (GBP)
Organisation British Society for the History of Science (BSHS) 
Department Darwin Trust of Edinburgh
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description EMBO Long Term Fellowship - Schiavone
Amount € 70,000 (EUR)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 06/2012 
End 05/2014
 
Description EMBO Long Term Fellowship - Yulzary
Amount £70,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Learned Society
Country European Union (EU)
Start 07/2010 
End 06/2012
 
Description Fanconi Anemia Research Fund Project Grant
Amount £75,000 (GBP)
Organisation Fanconi Anemia Research Fund (FARF) 
Sector Charity/Non Profit
Country United States
Start 06/2011 
End 06/2013
 
Description Jurgen Manchot Stiftung Studentship
Amount £47,000 (GBP)
Organisation Jurgen Manchot Foundation 
Sector Charity/Non Profit
Country Germany
Start 10/2011 
End 09/2014
 
Description NPIF Rutherford Fund Fellowship - Crisp
Amount £158,819 (GBP)
Funding ID AS16 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 11/2017 
End 10/2020
 
Description Summer Scholarship - Traparic
Amount £1,320 (GBP)
Organisation Amgen Foundation 
Start 07/2018 
End 09/2018
 
Description Summer Studentship - Gilmartin
Amount £1,600 (GBP)
Organisation Biochemical Society 
Sector Learned Society
Country United Kingdom
Start 07/2016 
End 08/2016
 
Description Wellcome Trust TMAT Studentship
Amount £200,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description Balasubramanian 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Examining impact of G-quadruplex stabilising ligands on epigenetic instability, combining our cell line models with the expertise of our chemist collaborators, Professor Shankar Balasubramanian & Dr. Pierre Murat, in biophysics of G quadruplexes and synthesis of G quadruplex ligands
Collaborator Contribution Provided expertise in biophysics of G quadruplexes and compounds that can stabilise these structures
Impact Publication: Sarkies, P., Murat, P., Phillips, L.G., Patel, K.J., Balasubramanian, S. and Sale, J.E.¶ (2011). FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA. Nucleic Acids Research DOI 10.1093/nar/gkr868.
Start Year 2010
 
Description COMSIG consortium 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Named investigator on Wellcome Trust COMSIG consortium, which is a multi centre and multi disciplinary grant to investigate the origins of mutational signatures. We have provided constructs and scientific advice.
Collaborator Contribution Steve Jackson, Gurdon Institute and Michael Stratton, Sanger Centre run the project, providing personnel, grant administration, space and equipment.
Impact x
Start Year 2013
 
Description COMSIG consortium 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Named investigator on Wellcome Trust COMSIG consortium, which is a multi centre and multi disciplinary grant to investigate the origins of mutational signatures. We have provided constructs and scientific advice.
Collaborator Contribution Steve Jackson, Gurdon Institute and Michael Stratton, Sanger Centre run the project, providing personnel, grant administration, space and equipment.
Impact x
Start Year 2013
 
Description Caroline Dean 
Organisation John Innes Centre
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Studying the contribution of potentially structured DNA to the control of the FLC locus in Arabidopsis vernalisation
Collaborator Contribution Postdoc from the Dean lab embedded in my group for a year from March 1 2019
Impact n/a
Start Year 2018
 
Description Takeda 
Organisation University of Kyoto
Department Department of Radiation Genetics
Country Japan 
Sector Academic/University 
PI Contribution exchange of cell lines, data and ideas
Collaborator Contribution exchange of cell lines
Impact 16923963 17283053 18662998
 
Title ANGIOPOIETIN-2 SPECIFIC TIE2 RECEPTOR 
Description An Ang-2 specific ligand trap developed by directed evolution in a hypermutating cell line. A potential therapeutic with applications in cancer, inflammatory disease and sepsis. 
IP Reference WO2014096855 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact Too early to say
 
Title Antibody selection from hypermutating cell lines 
Description The discovery of cell lines undergoing constitutive immunoglobulin hypermutation & a method to select antibodies of a desired affinity from a pool such cells. Filed under: Sale, J.E., Neuberger, M.S. and Cumbers S.J. (2000). 'Method for generating diversity' PCT/GB99/03358 WO 00/22111 Medical Research Council, U.K. Sale, J.E. and Neuberger, M.S. (2001). 'Method for generating diversity' CIP. Medical Research Council, U.K. 
IP Reference EP2204447 
Protection Patent granted
Year Protection Granted 2010
Licensed Yes
Impact Licensed by MRC to Anaptys Inc., I understand.
 
Title Antibody selection from hypermutating cell lines 
Description The discovery of cell lines undergoing constitutive immunoglobulin hypermutation & a method to select antibodies of a desired affinity from a pool such cells. Filed under: Sale, J.E., Neuberger, M.S. and Cumbers S.J. (2000). 'Method for generating diversity' PCT/GB99/03358 WO 00/22111 Medical Research Council, U.K. Sale, J.E. and Neuberger, M.S. (2001). 'Method for generating diversity' CIP. Medical Research Council, U.K. 
IP Reference US2008193979 
Protection Patent granted
Year Protection Granted 2008
Licensed Yes
Impact Licensed by MRC to Anaptys Inc., I understand.
 
Title Angiopoieitin 2 ligand trap 
Description In collaboration with Prof Nick Brindle (University of Leicester), who joined the lab as a sabbatical visitor for a year, we used directed evolution in DT40 cells to evolve a ligand trap for Angiopoietin II (see Brindle, Sale et al JBC 2013 PMID: 24106271). Nick is continuing to study and develop the molecule in mouse models in Leicester, and we are seeking to further develop it with the help of MRCT. A patent application was submitted by MRCT in Dec 2012. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Initial development
Year Development Stage Completed 2012
Development Status Actively seeking support
Impact This is the first ligand trap for Ang II and as such has great therapeutic potential in inflammatory and malignant diseases. 
 
Description Big Biology Day 
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 Leticia Koch-Lerner participated in the Big Biology Day (13.10.18) Hills Road Sixth Form College with the activity "Synthetic biology: Create a New Protein" (design and create your
own model protein in clay using concepts from synthetic biology)
Year(s) Of Engagement Activity 2018
URL http://www.hillsroad.ac.uk/college-life/events/2018/10/13/default-calendar/big-biology-day
 
Description Cambridge Science Festival 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Guillaume Guilbaud and Leticia Koch-Lerner contributed to the Cambridge Science Festival (17.03.18 Guildhall Cambridge) in the activity "Worms are cleverer than you think" (identification of different types mutated worms by observation of live worms under the microscope).
Year(s) Of Engagement Activity 2018
URL https://www.sciencefestival.cam.ac.uk
 
Description LMB Open Day 2013 - Chairing Public Lectures 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talks were followed by questions and positive comments on feedback forms about value the research and public's interest in it

Requests for similar events. Better levels of understanding (from feedback forms).
Year(s) Of Engagement Activity 2013
 
Description LMB Open Day 2013 - Group members assisted with science demonstrations 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Demonstrations promtped questions and requests for further information

Better understanding among public about the work of the LMB
Year(s) Of Engagement Activity 2013
 
Description LMB Open Day June 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Members of the sale lab helped prepare and deliver activities in the 'Biological Building Site' area. Feedback from children and adults was very positive. Children reported 'now I want to be a scientist.'
Year(s) Of Engagement Activity 2017
 
Description Pint of Science 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Two lab members (Guillaume Guilbaud and Leticia Koch-Lerner) contributed to the organisation and running of three 'Pint of Science' events on 'My Body' 14-16.5.18 at 'The Maypole'.
Year(s) Of Engagement Activity 2018
URL https://pintofscience.co.uk