Reconstitution of nucleotide excision repair at the single molecule level in vitro and in vivo

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Simply stepping outside on a sunny day exposes the skin to enough ultraviolet radiation (UV) to cause blistering and the formation of cancerous tumours. Why this doesn't occur is due to enzymes present in every cell that scan DNA for damage and then initiate repair. Xeroderma pigmentosum (XP) is one of a number of diseases caused by deficiencies in this repair pathway and for individuals with XP this leads to skin blistering, cancer and neurological dysfunction. A complete lack of these nucleotide excision repair (NER) enzymes is lethal. Because most organisms are exposed to UV, this mechanism of DNA repair is conserved across all forms of life. In humans over 30 enzymes are involved in NER, whereas in bacteria only 6 enzymes are required. Therefore understanding NER at the simpler bacterial level will provide insight into the human equivalent. Despite decades of research into NER there is surprisingly little known about the precise details. The components are well-established but how they work together is still uncertain. The main aim of our work is to understand how the bacterial system works as a whole, but still at the molecular level. This is important because the classical approach of studying individual components may miss the formation of enzyme complexes or overstate the importance of individual components. This is very complex and therefore we study single molecules to simplify the system. We aim to directly watch complexes forming, their mechanisms of damage location and the recruitment of other components. These are all physical concepts; a protein has to search through a sea of undamaged DNA to find the lesion, somehow it must communicate with other proteins to signal that it has achieved this goal and then organise these other proteins onto the site of damage. Only through single molecule imaging of a complex mixture of components can we get a true picture of how DNA is repaired. To take this further we are also proposing to image these processes in live bacteria. We will use cutting edge techniques to isolate single molecules within cells and study how they behave alone and with each other. This is immensely exciting; the prospect of visualising single molecule processes in their native environments is a very new field of study. These combined approaches will offer a complete view of how DNA repair occurs in vitro and in vivo.Not only will this project improve our understanding of bacteria repair it will serve as a proxy for understanding how proteins interact with DNA more generally. There is a gap in our toolset from cell biology to single molecule imaging that we will fill during this project. Therefore the tools and techniques that we develop will find application across a wide range of problems. Ultimately, the knowledge gained from this study will inform studies of human equivalent systems, such as XP. This will have considerable impact on the lives of individuals with this highly debilitating condition.