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

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.

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.

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?
Major 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.