Stress, DNA damage and disease

Lead Research Organisation: University of Wales
Department Name: UNLISTED

Abstract

The impact of defective DNA repair is seen in patients with xeroderma pigmentosum, who have very high incidences of skin cancer due to being unable to remove sunlight-induced DNA damage and in patients suffering with predispositions to colorectal and other cancers. Simply put, un-repaired DNA damage causes mutations which contribute to the incidences of genetic defects and to cancer. Hence DNA repair is crucial to reduce the incidence of harmful mutations.
This MRC co-operative award allows several research teams working on DNA repair, cell stress and cancer to benefit from core technologies that support all of them. One technology provides the ability to follow DNA repair proteins by tagging them with fluorescent factors to enable them to be detected in cells, so as we can see how they react to DNA damage. The other technology enables us to analyse DNA repair proteins via mass spectrometry to unravel their composition; this is the first step in understanding the structure of repair proteins so as we can determine how they interact with DNA in our cells to detect and remove DNA damage.

Technical Summary

Little is known as to how DNA excision repair functions in DNA packaged as chromatin, be it in active or repressed domains, or in telomeres. Large complexes metabolizing DNA, such as those involved in transcription, require an intricate series of events to function productively in this environment. It is now evident that for transcription, different chromatin remodelling events alter the structural organization of different parts of the genome, and that some regions are arranged in domains, where specific types of remodelling can predominate.
Large complexes involved in DNA repair require specific factors to operate in chromatin, and chromatin remodelling is intimately linked to the cancer process. How chromatin remodelling relates to repair, cell stress, cell ageing or tumourigenesis needs to be addressed. We wish to build on existing strengths in our areas of focus, especially nucleotide excision repair and mismatch repair so as to foster new collaborations to tackle these issues.

Publications

10 25 50
 
Description COMARE on Sunbed Legislation
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact We have advised that unmanned sunbeds are banned, that manned parlours are only available to those over 18 and that they are staffed by individuals who advise clientele with respect to the risks.
 
Description Chairman of UK Genome Stability Network
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact Provides a forum for training and development of UK based students and post docs working in the field of genome stability.
 
Description Training of UK based genetic toxicologists
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Continuous development for professional genetic toxicologists in UK industry.
 
Description Working Group for Improved Radiation Therapy
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact The working group aims to improve the delivery of radiotherapy in the UK.
 
Description CRUK Project award
Amount £250,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 03/2014
 
Description CRUK funding (Analysis of in vivo tumour suppression mediated through Lkb1 and Pten)
Amount £240,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2007 
End 06/2010
 
Description CRUK funding (Investigating the PI3 kinase and Ki-Ras pathway interactions in colorectal cancer: new models and drug validation)
Amount £220,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2010 
End 12/2013
 
Description CRUK funding (The Wnt pathway in colorectal disease: Validation of novel targets and intervention strategies and the refinement of existing models)
Amount £450,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2006 
End 09/2009
 
Description CRUk funding (Validating methylation sensing proteins as therapeutic targets)
Amount £1,700,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2008 
End 06/2013
 
Description EP/F040954/1 EPSRC Basic Technology Programme (Intracellular Biophotonic Nanoswitches)
Amount £1,423,468 (GBP)
Funding ID EP/F040954/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 09/2012
 
Description Epigenetics and Cancer
Amount £43,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2009 
End 11/2010
 
Description Infrastructure Award to Establish Structural Biology as a Discipline at Cardiff
Amount £60,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 10/2010
 
Description Investigating the immediate and therapeutic effects of MEK inhibition and P13K inhibition in a genetically relevant autchthonous model of human colorectal cancer
Amount £70,000 (GBP)
Funding ID PhD2009/L14 
Organisation Tenovus 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description MRC Research Grant (Development of new agents for colorectal cancer (CRC) using genetically defined autochthonous models)
Amount £400,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2009 
End 09/2011
 
Description MRC Studentship - Investigating the role of ABF1 binding in nucleotide excision repair
Amount £53,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2007 
End 10/2010
 
Description NISCHR Cancer Genetics BRU Award (The development of novel methods for translational studies in cancer genetics)
Amount £1,500,000 (GBP)
Organisation Health and Care Research Wales 
Sector Public
Country United Kingdom
Start 01/2011 
End 12/2014
 
Description Studentship - Determining the non-proteolytic function of a novel E3 ubiquitin ligase in NER
Amount £26,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2006 
End 10/2009
 
Description University of Cardiff College of Medicine, Lord Merthyr Scholarship
Amount £30,000 (GBP)
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 10/2011
 
Description VIP Award
Amount £35,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2006 
End 01/2007
 
Description Wales Gene Park
Amount £1,654,640 (GBP)
Organisation Health and Care Research Wales 
Sector Public
Country United Kingdom
Start 04/2007 
End 03/2010
 
Title Expression arrays for UV induced gene transcription 
Description Gene expression profiles for UV regulated genes by a novel E3 ubiquitin ligase 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2008 
Provided To Others? Yes  
Impact EMBO J 2006 Paper and PhD Thesis 2010 
 
Title GGNER Complex Function in Vivo 
Description Novel strains to investigate function of the GGNER proteins in yeast 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact Papers JBC - 18996839 EMBO J - 16675952 
 
Title GGNER Complex Purification and Characterisation 
Description Newly identified E3 ligase function of the GGNER complex 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2007 
Provided To Others? Yes  
Impact Papers EMBO J - 16675952 
 
Title Generated Series of Novel Mouse Models of Human Colorectal Disease 
Description We have generated a series of novel mouse models of human colorectal disease. These include mice mutant for Apc and Pten (2009), mice mutant for Lkb-1 (2009), mice mutant for K-ras and Apc (2007). All of these models, which are aimed at sequentially replicating the stages of human colorectal cancer have been provided freely to other research groups. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact Virtually all new activity in the group is based around the generation of these novel models, hence we have initiated collaborations as detailed in the collaborations section. Also new grant revenue as detailed in period was dependent upon the development of these models. 
 
Title Novel mammalian cell line for assessing the impact of drug transporters in anticancer drug resistance 
Description We have developed and characterised the nature of ABCG2-associated resistance to Hoechst 33342 and the model anticancer drug topotecan in a murine cell model with constitutive expression of side population characteristics. This is the first stable cell line for defining the 'side population' phenotype for incorporation into flow cytometric assays of stem cell populations. The details have been released in 2009 (PMID: 19802874) 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2007 
Provided To Others? Yes  
Impact Public disclosure only in 2009 (PMID: 19802874) 
URL http://europepmc.org/abstract/MED/19802874
 
Title Omics method for detection of DNA damage and repair 
Description Novel tools for detecting genome wide DNA damage and repair at high resolution throughout genomes 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact NAR Paper 2010 IP published 2009 Collaboration with Agilent Technologies and KTP awarded to develop the method for gentox testing. NISCHR BRU grant. 
 
Title ProgeniDB 
Description A novel cell lineage database for generation associated phenotypic behavior in cell-based assays. Public release 2007: PMID: 17387278 
Type Of Material Database/Collection of Data/Biological Samples 
Year Produced 2007 
Provided To Others? Yes  
Impact The database is no in use for developing cel-cycle models with systems biology groups in Warwick and Swansea. The core data has laid the foundation for a two-site EPSRC bid for platform funding. 
URL http://europepmc.org/abstract/MED/17387278
 
Title Stroboscopic fluorescence lifetime imaging 
Description We report a fluorescence lifetime imaging technique that uses the time integrated response to a periodic optical excitation, eliminating the need for time resolution in detection. This stroboscopic technique offers lifetime based imaging at video rates with standard CCD cameras and has application in probing millisecond cell dynamics and in high throughput imaging assays. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Has underpinned a PhD studentship and MRC discipline hopping Fellowship. Currently in use and development in concert with a newly established collaborative group with Swansea University MNC. 
 
Title Technology to examine DNA damage repair 
Description The method enables us to examine DNA repair for entire genomes on microarrays and to determine how chromosomes are modified to facilitate the functioning of repair. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact There is currently a patent in progress - patent number 0712584.2. 
 
Title Tools for analysing chip chip data for DNA damage induction 
Description Bioinformatic tools for normalising and peak calling for the detection of UV induced DNA damage by Chip CHip 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact NAR paper 2010 
 
Title Yeast Rad4 antibody, Yeast Rad33 antibody 
Description Novel antibodies against two key NER proteins 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact EMBO J Paper on Rad23/4 function in NER. 
 
Description A role for Mec1 in NER 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We examined this by first housing his PhD student, Michael Taschner, who worked with Yumin Teng (our MRC PD) to employ technology we had developed and then by analysing samples at both labs.
Collaborator Contribution Our collaborator, Jesper Svejstrup, had suspected that there was a role for Mec1 in NER.
Impact We showed that there was a role for Mec1 in phosphorylation in governing both TC-NER and GG-NER (see paper 18936173).
Start Year 2008
 
Description Biochemical Function of the Yeast GGNER Complex 
Organisation Case Western Reserve University
Department Department of Biochemistry
Country United States 
Sector Academic/University 
PI Contribution We uncovered the biochemical activities of the yeast GGNER complex in chromatin remodelling.
Collaborator Contribution Our collaborator trained one of our researchers in several key chromatin remodelling assays.
Impact JBC Paper 18996839 Work was also cited in a review by our collaborator
 
Description Generating Antibodies against Novel NER Components 
Organisation Enzo Life Sciences
Country United Kingdom 
Sector Private 
PI Contribution We identified a number of novel NER factors and are using the newly generated antibodies to discover the functions of these proteins.
Collaborator Contribution Enzo Life Sciences have generated a number of key peptide antibodies raised against several NER factors.
Impact EMBO J - 16675952 JBC Paper - 18996839
 
Description How Rad4 and Rad34 contribute to rDNA repair in relation to chromatin structure 
Organisation University of Sherbrooke
Country Canada 
Sector Academic/University 
PI Contribution We applied the technology that we had developed to examine NER and nucleotide resolution in the UV irradiated cell samples provided by our collaborator.
Collaborator Contribution Our collaborator, Antonio Conconi, developed the method to separate the repressed and active copies of the rDNA genes and sent us samples from UV irradiated cells.
Impact We were able to uncover exactly where there were separate roles for the Rad4 and its homologous Rad34 NER protein in NER (see paper 18936173).
Start Year 2007
 
Description NER and chromatin modifications in subtelomeric regions of yeast 
Organisation University of Nottingham
Department School of Molecular Medical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We modified the strains substantially to examine how NER operated on the URA3 gene when relocated to repressed versus transcriptionally active subtelomeric regions.
Collaborator Contribution Our collaborator, Ed Louise, provided the strains.
Impact We uncovered a role for Sir2 in suppressing UV induced H3 acetylation. All work was undertaken at Cardiff University by Yachuan Yu (MRC PD) and A Irizar (MRC PhD). A paper has been written and submitted.
Start Year 2006
 
Description NER, transcription and histone modifications at the PHO5 locus of yeast 
Organisation University of Oxford
Department Weatherall Institute of Molecular Medicine (WIMM)
Country United Kingdom 
Sector Public 
PI Contribution We used the strains provided to examine NER in exquisite detail at the PHO5 regulatory region under varying transcriptional states.
Collaborator Contribution Our collaborator, Peter McHugh, provided the strains and made substantial intellectual contributions to the project.
Impact We have discovered that despite the absence of nucleosomes in the PHO5 promoter transcription can inhibit NER in certain circumstances. We are currently examining the reasons for this.
Start Year 2007
 
Description Roles of Rad16 in modifying chromatin structure for NER 
Organisation University of California, San Diego (UCSD)
Department Department of Pathology
Country United States 
Sector Academic/University 
PI Contribution My research team set the ground work for this collaboration by identifying a role for Gcn5 in efficient NER, by developing the assays we employed to examine NER at nucleotide resolution an by showing that hyperacetylated regions did not require Rad16 for NER.
Collaborator Contribution My collaborator Simon Reed and myself have identified a key role for Rad16 in facilitating post UV histone acetylation to facilitate efficient NER. Rad16 determines the recruitment post UV of the histone acetyltransferase Gcn5 which is a key factor in enabling efficient NER in parts of the yeast genome.
Impact PNAS publication (pubmed ref) EMBO Reports last year (ref) Three papers in draft form 1 - The role of Rad16 in facilitating histone acetylation post UV submitting to Molecular Cell 2 - The role of histone H3 and H4 acetylation post UV in facilitating NER in subtelomeric regions (under revision for Nucleic Acids Research) 3 - A method to examine DNA damage at high resolution in entire genomes (submitted to Nature Methods)
 
Description Studies on Global Genome Repair in Yeast 
Organisation University of California, San Diego (UCSD)
Department Department of Pathology
Country United States 
Sector Academic/University 
PI Contribution Certain DNA repair assays for interpretation of specific biochemical data.
Collaborator Contribution Our research demonstrated specific biochemical activities of the GGNER complex which were then investigated further by examining in vivo DNA repair assays.
Impact EACR Cancer Researcher Award International Patent granted Publications 18996839 17150417 16675952
 
Description The Role of the Proteasome in Nucleotide Excision Repair 
Organisation University of Texas
Country United States 
Sector Academic/University 
PI Contribution We provided all of the expertise and reagents necessary for investigating the connection with nucleotide excision repair.
Collaborator Contribution Our collaborator provided a number of key strains and reagents necessary for our investigation into the role of the proteasome in NER.
Impact EMBO J - 16675952 Roads to Rome review - 17150417
 
Title Microtrench - a novel device for the analysis of cell proliferation 
Description Published Earliest priority date 29/01/2008 International filing date 29/01/2009 Field of the Invention: Cell biology and drug screening. The invention relates to the use of a cell culture microtrench having dimensions which allow attachment of cells to the surface of the trench and constrain the location and behaviour of cells for analytical advantage. The invention also relates to such a microtrench, arrays of such trenches with varying dimensions to provide for selection of optimal assay characteristics for a user-defined purpose, and arrays of trenches with the same dimensions to facilitate the scaling up and efficiency of a given assay. The trenches and/or arrays can be readily incorporated into convenient methods of cellular analysis in research, diagnostics and screening assays. WO2009095666 (A1) 
IP Reference WO2009095666 
Protection Patent granted
Year Protection Granted 2009
Licensed Commercial In Confidence
Impact Expolitation partnership currently underway
 
Title Technology to examine DNA damage and repair 
Description The method enables us to examine DNA damage and repair for entire genomes on microarrays and to determine how chromosomes are modified to facilitate the functioning of repair. 
IP Reference GB2463197 
Protection Patent granted
Year Protection Granted 2010
Licensed No
Impact The clinical sector have noted that our technology can be applied to examine the reaction and fate of anti cancer therapies such cisplatin in patients undergoing therapy.
 
Description Hosting Visits - Cancer Charity Supporters 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact We hosted visits by cancer charity supporters so that the could see how their donations were being put to work.

The most positive outcome has been direct financial support either to the University or to a Charity.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Lecture - The RAFT Institute, Mount Vernon Hospital 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Health professionals
Results and Impact I was invited to present our new technology for examining DNA damage at high resolution in entire genomes with a view to setting up collaborations to examine UV damage in skin tissue.

None as yet.
Year(s) Of Engagement Activity 2009
 
Description Permanent AudioVisual Display at "Frontiers "- at National Waterfront Museum, Swansea 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Development of a permanent dispaly for the museum as an effective method of reaching out to thousands of visitors - allowing them to investigate Welsh upcoming innovations in the Frontiers gallery of the national Museum - in particular cancer diagnostic technologies. http://www.museumwales.ac.uk/en/swansea/frontiers/

A highly effective method of reaching out to thousands of visitors, school parties and officials.
Year(s) Of Engagement Activity 2007,2008,2009
URL http://www.museumwales.ac.uk/en/swansea/
 
Description Podcasts for CRUK 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact The first podcast was regarding a publication that appeared in Nature Genetics about the identification of a novel therapeutic target for colorectal cancer. The second was regarding the award of the new Cardiff CRUK cancer centre.

We did receive some direct public donations, we were also featured on both ITV and BBC news.
Year(s) Of Engagement Activity 2007,2009
 
Description Policy - COMARE Report on Sunbed Legislation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Policymakers/politicians
Results and Impact Here I used our findings to suggest further investigations into how risk of radiation exposure is measured and perceived.

Two high profile interviews on BBC breakfast television to increase public awareness of the risks associated with sunbed use and sunbathing.
Year(s) Of Engagement Activity 2008
 
Description Public Lecture - Royal Institute of South Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact I described how DNA repair is required to reduce cancer and mutational risk, how one can study this highly conserved process in model organisms. I then went on to outline how our contributions employing yeast as a model have unravelled how the genome compacted as chromatin is accessed for repair and restored to its pre-damaged status. I ended by describing the implications of our research on human health and cancer risk.

Considerable public interest and many questions.
Year(s) Of Engagement Activity 2008
 
Description Public Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Public lectures to local groups including various charity organisations, e.g. Rotary Club.

Donations to Cancer Research Wales.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Visit by Wales Assembly Members - inc First Minister 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Policymakers/politicians
Results and Impact Hosted an informal visit by the First Minister to inform him of Cancer Research activities in Biosciences and hosted a second informal visit by the First Minister to introduce the Cardiff CRUK Cancer Centre and the WAG funded PET centre.

Continued interest/discussion with WAG re-enhancement/investment in cancer activity within Cardiff.
Year(s) Of Engagement Activity 2007,2009
 
Description Website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Published on line lay information for research undertaken in the group.

Invitations to speak at a number of charity fund raising events including Rotary Club, Cancer Research Wales and Probus.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010