Nucleotide Excision Repair - Lighting up a Dark Pathway

Nucleotide Excision Repair (NER), also known as "Dark Repair", is a DNA repair pathway essential for all forms of life including humans. Mutations in this pathway lead to devastating diseases such as xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) with elevated cancer levels and premature ageing. NER is highly complex, involves many different proteins, and is still not well understood. There are particular hurdles in the study of dynamic processes such as NER that involve transient interactions of large protein and DNA complexes. Advances in protein production and in new techniques to study molecules one at a time (thus avoiding averaging effects) have opened the door to new ways to study and understand these complex processes, and we now have all the tools we need to study NER in detail. By "lighting up" individual protein complexes we can follow their behavior in real time, and their interactions with other DNA and protein species. Recent advances in structural biology techniques will allow these large protein complexes to be studied at a molecular level. This work will advance technology and lead to a step change in our fundamental knowledge of a key aspect of our biology, essential for healthy ageing.

The Nucleotide Excision Repair (NER) pathway is unique in its ability to detect and remove a wide range of distorting DNA lesions, and is thus essential for genome integrity and healthy ageing. This important pathway is still poorly understood at a molecular level due to technical challenges that we have now overcome. NER requires over 20 proteins, including the 10-subunit Transcription Factor IIH (TFIIH), which collaborate in the key steps of repair: damage detection, DNA opening, proofreading, incision and repair synthesis. Mutations of NER proteins in humans can lead to high levels of DNA damage and progeria (premature ageing). The NER pathway is complex and dynamic, involving multiple transient protein:protein and protein:DNA interactions, making its study difficult-to-impossible by traditional methods. We have developed new protein expression, labelling and assay strategies for the human NER pathway. We will use a range of techniques including single molecule fluorescence, mass spectrometry, cross-linking and electron microscopy to dissect each step of the NER pathway. This will achieve a step-change in molecular understanding of each stage from recruitment and enlargement of the repair bubble to damage verification and incision. This work is at the cutting edge of molecular studies of complex cellular processes, and given the ubiquity of DNA distortion by proteins in molecular biology, the work will have a major impact on a very broad field.
The project is in the following strategic priority areas: healthy ageing across the life course; international partnerships; technology development for the biosciences.

Public Engagment
The public will benefit from outreach activities linked to this application. The PI and Co-I are committed to activities supporting the public understanding of science - evidenced by a track record of engagement with Schools, public lectures and science festivals such as the Cheltenham science festival. School pupils will also get an opportunity to attend the laboratory and gain work experience (average of 1 student per year in the last 3 years). The team employed on the grant undertake to prepare and deliver an exhibit for science festivals. This work will expose the public, particularly young people, to exciting science and enhance their understanding of the importance of DNA Repair. The team will also take part in the Wellcome Trust-funded Cell Block Science programme, which takes science learning into Scottish prisons.
A key part of our plan for public engagement is the production of a short animated film that will explain the concepts of DNA damage and repair to a lay audience. There are many misconceptions in the general population around DNA damage, which is often considered as a rare phenomenon that always leads to cancer. We aim to improve public awareness in this area significantly.
Academic (educational) impact
Working with Vivomotion, we will produce a short animated movie to illustrate the process of Nucleotide Excision Repair at a level of detail suitable for academic audiences - including undergraduate teaching. This movie will be made freely available for distribution to maximise its impact.
Research and professional skills
The PDRA employed on the grant will receive extensive training in a variety of disciplines spanning microbiology through biochemistry to biophysics. They will have access to the award winning courses run by the University of St Andrews, which aim to provide a wide variety of transferrable skills. By the end of the project they will have acquired the "Passport to Research Futures" a structured development programme tailored by St Andrews revolving around career planning, professional development and employability.
Economic and Societal Impact
The NER pathway is an attractive target for drug development in the area of cancer therapy, as NER can reverse the effects of widely used chemotherapeutic agents that damage DNA. Two compounds, spironolactone and triptolide which both target TFIIH, are in clinical use (see Is TFIIH the new Achilles heel of cancer cells?, Berico and Coin 2017 http://dx.doi.org/10.1080/21541264.2017.1331723 for more information). This field has been hampered by the lack of a robust assay for TFIIH activity. We will therefore explore with partners in industry or academia the use of our reagents and assays for inhibitor screening. We will work with our dedicated Business Development Manager to explore all opportunities for the maximisation of applied outcomes arising from this research.