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

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Barnett JT (2019) Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Van Houten B (2018) Single-cell mutagenic responses and cell death revealed in real time. in Proceedings of the National Academy of Sciences of the United States of America

 
Description As a result of this work we have been able to uncover the mechanism of DNA repair with a new level of accuracy. We have implemented a novel high speed and spatial resolution approach to study the role of the complexes formed in vivo. This has, for the first time, enabled us to link DNA repair to replication, this has substantial impact for our understanding of DNA repair, which in turn could affect how we develop anti-microbial compounds in the future.
Exploitation Route We expect that this improved understanding of DNA repair will be useful for the design of anti-microbials. Currently, there has not been a new class of such drugs for ~40 years, and it is imperative that such basic research is performed in order for the development of new drugs.
Sectors Healthcare

 
Description The outcomes from this project have begun to be exploited towards the development of new anti-microbials. This is a direct outcome from the reagents created during this project.Therefore, there are improved health and life quality impacts.
First Year Of Impact 2020
Sector Healthcare
Impact Types Societal

 
Description Squeezing the most out of DNA; developing nanoconfinement tools to study DNA repair
Amount £90,668 (GBP)
Funding ID APX\R1\191100 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2021
 
Description Studying eukaryotic NER 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution We provided the single molecule expertise and access to equipment.
Collaborator Contribution Access to the protein raw materials, including specific mutants and biochemical expertise.
Impact This project has involved the single molecule biophysics from the Kad lab and protein chemistry and biochemistry from the Kisker lab. We have presented data at meetings and also have submitted a paper for publication.
Start Year 2016
 
Description Understanding nucleotide excision repair with Ben Van Houten 
Organisation University of Pittsburgh
Country United States 
Sector Academic/University 
PI Contribution We have been working with Ben Van Houten to develop a deeper understanding of DNA repair.
Collaborator Contribution The Van Houten group offers expertise in the area and also a number of plasmid constructs and damage substrates. In addition, previously unpublished data from the Van Houten group is shared with us.
Impact This relationship has led to the publication of several articles, development of new technologies and international recognition of our work.