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

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.

Nucleotide excision repair (NER) specialises in the removal of bulky DNA lesions particularly those induced by UV exposure. Six enzymes work together to locate and repair damage in the prokaryotic system versus the 30-plus enzymes involved in mammalian NER. Both systems share a common mechanism: the lesion is located, an endonuclease is recruited and the DNA nicked either side of the damage. Following this, the damage containing oligonucleotide is released and DNA resynthesized. The components of the bacterial system are well-established; however how they work together still remains unclear.

In this project we propose to use single molecule imaging on our unique DNA tightrope platform where single DNA molecules are suspended between surface immobilised beads, to investigate how bacterial NER works as a whole. We have developed a six-colour imaging system specifically for this investigation capable of simultaneously visualising all the repair components at once, with high time resolution. By differentially tagging each component and characterising the complexes that form on DNA tightropes we can assign roles to these complexes in repair. In parallel, we will use cutting-edge stroboscopic and super-localisation techniques to measure complex formation and action within living bacteria, at the single molecule level. This will provide confirmation of the complexes formed in vitro. By studying repair in vitro and in vivo simultaneously we can use results from one platform to direct experiments on the other.

The results from this research will find application in a number of areas. The principle users of our findings will be the academic community and those involved in developing antimicrobial technologies. As a consequence, this work is relevant to the BBSRC strategic priority areas of: Systems approaches to the biosciences, Combatting antimicrobial resistance and technology development for the biosciences.

We are implementing a newly developed technology to permit more complex biological systems to be imaged in real time. Therefore there will be beneficiaries across a number of fields that use imaging technologies, whether within the biosciences or beyond. The use of this technology will be both at the single molecule level in vitro and in cells. Furthermore understanding complex systems using an imaging based approach requires multiplexing in real time. The biosciences are moving in the direction of whole systems studies, and the combination of techniques used here strongly address this. By having tools to enable direct investigation of such interactions with the capability of super-localisation this goal can be more easily realised.

Defects in NER cause severe human diseases including xeroderma pigmentosum, trichothiodystrophy and cockayne's syndrome. To uncover principles that underpin these complex human disease systems we will study the equivalent DNA repair process in prokaryotes. Also, since the mechanism of DNA repair is shared between prokaryotes and eukaryotes, but the protein homology is substantially distinct, a fuller understanding of NER offers a mechanism for the development of antimicrobial drugs specifically targeted to this crucial pathway. To develop interactions with industry we will use with the Research Services and the Innovation & Enterprise office, for the latter Biosciences has an embedded academic for improved access.

Real time imaging is used across disciplines ranging from number plate detection to airport security, having the tools present to image multi-spectrally in real-time will find beneficiaries in these areas. To ensure that the wider public is aware of our work we will publicise our findings through the University of Kent's Press Office looking for publication in local and national technical and non-technical journals.
Single molecule techniques are relatively new and are becoming more widely exploited from the level of basic research to applied science. An example is genome sequencing which uses single molecule technologies in next generation sequencers. The staff involved in this research proposed here will receive specific project related training across a wide range of disciplines, offering an excellent opportunity to learn cutting-edge skills. In addition they will learn transferable skills from conference networking, giving seminars, and writing reports.

In this proposal we are seeking to understand a complex process with single molecule resolution. Investment in such research will enable the UK to remain internationally competitive as a knowledge economy. Presently, many other nations particularly Holland, Germany and the USA are making substantial investments in this area.