Determining how global genome nucleotide excision repair promotes efficient removal of DNA damage from chromatin

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

Contained within each of our cells is the coded information necessary for life. The information is stored in a compartment of the cell called the nucleus which contains a large molecule with a remarkable structure called deoxyribonucleic acid - DNA. The information within the DNA is divided into units called chromosomes which are further subdivided into genes. DNA is packaged into chromatin in order to compact the genetic material in the nucleus and the sum of all the genetic material of an organism is referred to as its genome. It might be anticipated that life's coded information would be extremely stable and resistant to change, since errors in the code could have serious consequences. On the other hand, organisms are able to adapt to changes in their environment by virtue of the genetic variation within the population caused by alterations in the genetic material of individuals. This process is known as evolution. In the human population a lot of genetic variation is the result of 'reshuffling' of the genes during sexual reproduction. However, DNA can also be altered by normal processes operating within the cell, as well as physical or chemical damage from the environment, including ultraviolet radiation from sunlight. The DNA in each of our cells is continuously damaged by such agents. If left unchecked this would quickly erode the genetic information, since copying damaged DNA when cells divide can permanently alter the genetic code - a process called mutation.

Over time, a variety of different DNA repair pathways have evolved which serve to prevent this from occuring. Collectively these are fundamental to the stability of the genome. People who inherit defects in the genes controlling these DNA repair pathways are more likely to suffer from certain cancers and other diseases. Our research aims to understand how one of these processes, nucleotide excision repair [NER] operates. People with defects in genes regulating this process suffer highly elevated levels of skin and other cancers. Molecular studies have revealed how defects in different genes involved in the process can result in a number of clinically distinct diseases. Much of our knowledge has come from the study of NER in a variety of different organisms. We study NER in bakers' yeast. Amazingly, the mechanism in yeast is remarkably similar to that in human cells, underlining the fundamental significance of this process.

The work described in this proposal aims to help us understand how the NER process is organised in the genome and how lesions are removed from chromatin following UV induced DNA damage. It is emerging that the sensors of DNA damage in chromatin are playing an important role in how DNA repair is regulated in the cell. Research into DNA repair has entered a new phase of discovery, revealing how the various DNA repair mechanisms are controlled in response to DNA damage and how the pathways are integrated with one another. In addition to improving our understanding of the molecular basis of human disease syndromes, novel synthetic genetic interactions between the pathways are identifying new targets for novel and improved cancer treatments. At present our knowledge of how the NER pathway operates in chromatin and how this process is regulated lags behind our knowledge in other repair pathways. The work carried out in this proposal will significantly improve our knowledge in this area providing significant insight for further human studies.

Technical Summary

The main objectives of the work are to determine how the GG-NER process is organised and initiated throughout the genome in response to UV damage, and to determine how the GG-NER complex functions during chromatin remodeling to promote efficient repair in the genome.

We will use chromatin immunoprecipitation on microarrays [ChIP-Chip] to measure DNA repair factor binding throughout the yeast genome both before and after UV. We will also use this approach to measure epigenetic changes in the nucleosomes of chromatin in response to UV damage. Next generation sequencing on the ABI / Illumina platforms will be used to examine nucleosome mapping/chromatin remodeling in response to UV damage. We have direct access to the ABI SOLiD 5500 next generation sequencing system available via the Wales Gene Park and supported by a NISCHR funded BRU post in my laboratory. We have access also to the Illumina platform via our collaborator T. Owen-Hughes.

We have developed new technology to examine the frequency of DNA damage at high resolution throughout entire genomes (Teng, et al 2011 A novel method for the genome-wide high resolution analysis of DNA damage. Nucleic Acids Research, January; 39(2). The method was developed employing S.cerevsiae as a model organism. Via our approach we can measure DNA damage and its repair in the entire S.cerevisiae genome. This method allows us to determine how DNA damage is repaired in the context of chromatin. We will relate the repair events to changes in chromatin that facilitate DNA repair such as covalent histone modifications and chromatin remodeling activities as described in the proposal.

Biochemical studies on the in vitro activities of the GG-NER complex will involve the purification of native and recombinant proteins from yeast and bacteria. The biochemical activities of these proteins will be analysed using the in vitro experiments described in the case for support.

Planned Impact

Who will benefit?
Potential beneficiaries include the pharmaceutical, cosmetic, chemical and agribio sectors.
The research conducted will provide important information on the connection between the induction and repair of DNA damage and related changes in the epigenome. This has implications for the development of a novel method for genotoxicity testing we are working on with Agilent Technologies, for use in the above industries.

Commercial potential:
CU has a strong record of successful intellectual property exploitation. We have a dedicated office with 212 patent applications in biomedicine. In 06-07 it received a licensing income of £1.5M and ranked in the top 7 UK universities for this. CU has a contract with Biofusion, who ring-fenced £8.2M to invest in spin-out companies.
The technology developed to measure DNA damage throughout genomes which we will employ here, resulted in a patent being filed. A British Initial Patent application was filed at the UK patent office on 28/06/07. It entered the international PCT phase in 06/08. patent No. 0712584.2.

This patent has resulted in a KTP award with Agilent to develop the technology for genotoxicity testing. The Technology Strategy Board [TSB] used this application as an example to attract the MRC as a funder of the KTP and other TSB schemes. Agilent's Life Science Group has identified the toxicology market worldwide (estimated at £5 billion in 2011) as an important strategic growth opportunity for its business, as a major vendor of instrumentation and reagents for genomics, metabolomics and proteomics. In particular, it has identified three priority segments within this market: genotoxicity, developmental toxicity and systemic toxicity; in which investment in R&D, both internal, and through external collaborations with strategic partners, will be made. The KTP project with the Cardiff knowledge-base partner provides Agilent with a novel microarray based genotoxicity testing solution.

How will they benefit?
or regulatory changes are being introduced during the next two years with respect to genotoxicity testing, particularly in Europe. The development of novel improved genotoxicity testing methods is a major challenge. This will allow more accurate testing of novel and existing classes of drugs, chemicals cosmetics and foods in the industries mentioned above. Through an existing Knowledge Transfer Partnership with Agilent Technologies we are actively developing our technology for genotoxicity testing with end users in the chemical and pharmaceutical sectors.

Publications

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BenKetah A. (2013) Defining and targeting differentiation pathways in non-melanoma skin cancer in JOURNAL OF INVESTIGATIVE DERMATOLOGY

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Colmont CS (2014) Human basal cell carcinoma tumor-initiating cells are resistant to etoposide. in The Journal of investigative dermatology

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Colmont CS (2013) CD200-expressing human basal cell carcinoma cells initiate tumor growth. in Proceedings of the National Academy of Sciences of the United States of America

 
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 EEMS Committee member
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
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 Commonwealth Scholarship and Fellowship Plan
Amount £114,400 (GBP)
Organisation Government of the UK 
Department Commonwealth Scholarship Commission
Sector Public
Country United Kingdom
Start 01/2015 
End 01/2018
 
Description Feasibility Grant (Innovate UK/Unilever)
Amount £75,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 03/2015 
End 03/2016
 
Description Feasibility grant
Amount £240,000 (GBP)
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2015
 
Description Studentship (BBSRC/AstraZeneca)
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2020
 
Title Bioinformatic Tools 
Description Normalisation of Chip/chip data permitting qunatitative analysis of this data type 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Trialing the algorithms for inclusion in Agilent Technology's GeneSpring bioinformatic platform 
 
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 2014 
Provided To Others? Yes  
Impact NAR paper 2010 IP published 2009 Collaboration with Agilent Technologies and successful applications for KTP to develop the method for gentox testing and NISCHR BRU grant awarded. 
 
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 Technology assay or reagent 
Provided To Others? No  
Impact NAR paper 2010 
 
Title Chip-Chip data for DNA repair factors 
Description The rates at which lesions are removed by DNA repair can vary widely throughout the genome with important implications for genomic stability. We measured the distribution of nucleotide excision repair (NER) rates for UV induced lesions throughout the yeast genome. By plotting these repair rates in relation to all ORFs and their associated flanking sequences, we reveal that in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organised within the genome. We compared genome-wide DNA repair rates in wild type and in RAD16 deleted cells, which are defective in the global genome-NER (GG-NER) sub-pathway, demonstrating how this alters the normal distribution of NER rates throughout the genome. We examine the genomic locations of global genome NER factor binding in chromatin before and after UV irradiation, and reveal that GG-NER is organized and initiated from specific locations. By controlling the chromatin occupancy of the histone acetyl transferase Gcn5, the GG-NER complex regulates the histone H3 acetylation status and chromatin structure in the vicinity of these genomic sites to promote the efficient DNA repair of UV induced lesions. This demonstrates that chromatin remodeling during the GG-NER process is organized into domains in the genome. Importantly, we demonstrate that deleting the histone modifier GCN5, an accessory factor required for chromatin remodeling during GG-NER, significantly alters the genomic distribution of NER rates. These observations could have important implications for the effect of histone and chromatin modifiers on the distribution of genomic mutations acquired throughout the genome. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Invited review for Bioessays 
URL http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4641/?query=E-MTAB-4641.
 
Description Advancing the development and application of non-animal technologies 
Organisation Innovate UK
Country United Kingdom 
Sector Public 
PI Contribution Advancing the development and application of non-animal technologies
Collaborator Contribution Advancing the development and application of non-animal technologies
Impact Advancing the development and application of non-animal technologies
Start Year 2015
 
Description Advancing the development and application of non-animal technologies 
Organisation Unilever
Department Unilever Research and Development
Country United Kingdom 
Sector Private 
PI Contribution Advancing the development and application of non-animal technologies
Collaborator Contribution Advancing the development and application of non-animal technologies
Impact Advancing the development and application of non-animal technologies
Start Year 2015
 
Description Determining the mechanism of insertional mutagenesis caused by CRISPR/Cas9 genome editing 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Collaborator Contribution Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Impact Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Start Year 2016
 
Description Determining the mechanism of insertional mutagenesis caused by CRISPR/Cas9 genome editing 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC)
Country United Kingdom 
Sector Public 
PI Contribution Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Collaborator Contribution Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Impact Investigation of the DNA repair mechanisms driving CRISPR/Cas9 induced on and off-target strand break repair.
Start Year 2016
 
Title Genome wide assay for measuring DNA damage 
Description Genome wide assay for measuring DNA damage 
IP Reference WO2009001111 
Protection Patent granted
Year Protection Granted 2008
Licensed No
Impact Partnership with Agilent technologies
 
Title Genomic tools for measuring DNA damage 
Description Using Chip-chip/Chip-Seq methods to detect DNA damage [and repair] throughout genomes. Is being applied to genotoxicity testing and patient stratification of chemo drugs. Funded by: KTP [TSB/Agilent Technologies] NISCHR Cancer Genetics BRU CRUK Centre Clinical PHD fellow BBSRC/GSK Case award 
Type Support Tool - For Medical Intervention
Current Stage Of Development Initial development
Year Development Stage Completed 2012
Development Status Under active development/distribution
Impact Still being developed for different applications. Genotoxicity testing/stratified medicine 
 
Title Sandcastle - software for analysing Chip-chip datasets 
Description software written in R environment for the analysis of ChiP-Chip datasets 
Type Of Technology Software 
Year Produced 2014 
Open Source License? Yes  
Impact TSB industry partner grant with Unilever. Feasibility study for new gentox testing assays. 
 
Description Conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentations at national and International conferences on DNA Repair and Genome Stability and Genetic Toxicology in Holland, Denmark, Cambridge, France, Prague.

Webinars for Agilent Technologies and Thomson Reuters - International coverage
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL http://stateofinnovation.com/researcher-highlight-series-simon-reed?word=reed
 
Description Public Lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Public lectures to local groups including various charity organisations, eg Rotary Club.

Donations to Cancer Research Wales.
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Schools
Results and Impact Careers talks and lab visits recruitment of 6th formers to Cardiff University.

lab visit
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Website - Personal Research 
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 Publushed 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