Structural Mechanisms of Nucleotide Excision Repair

The genetic information in our cells is encoded on DNA, which is continuously exposed to sources of DNA damage. These can be of external origin, such as radiation and toxic chemicals, or originate from cellular processes that act on DNA. If unrepaired, such damage may lead to cell death, mutations, or human diseases including cancer. Therefore, our cells employ a number of molecular pathways to repair damaged DNA. I aim to study one of these pathways, called nucleotide excision repair, which serves to repair DNA damage induced by UV-light or reactive chemicals. Once damage is detected, DNA repair factors are recruited to the site of damage. Subsequently, these repair factors unwind the DNA double helix and cut out the piece of DNA that contains the lesion. Finally, the patch is fixed by synthesis of new, undamaged DNA.

By studying the three-dimensional structures of the molecules that participate in nucleotide excision repair, I aim to understand the mechanisms by which this pathway works, how it is regulated, and how mutations found in disease can lead to dysfunction of the pathway. The first step towards these goals is to recombinantly express the numerous components of the pathway, such that substantial amounts of highly purified material, which can also harbour engineered mutations if required for certain experiments, can be obtained. I will then apply a method called cryo-electron microscopy to undertake structural studies of the nucleotide excision repair pathway. This technique can resolve the molecular structure of biological molecules (such as proteins and nucleic acids) in such detail that three-dimensional models containing the positions of all the atoms in the molecular assembly can be constructed. These models help us understand how the molecular complexes involved in nucleotide excision repair perform their function. Applying these same methods to molecular complexes that harbour mutations will allow us to understand how human disease mutations interfere with the function of nucleotide excision repair. Together with my collaborator Prof. Wojciech Niedzwiedz (ICR), I will then use the insights obtained from these structural studies to conduct functional analysis of nucleotide excision repair complexes to place our findings in the biological context of the living cell. If we understand such DNA repair pathways in sufficient detail, we might be able to influence them to prevent or more efficiently cure disease.

The genomes of our cells are constantly exposed to DNA-damaging agents and processes. To preserve genomic integrity and cellular function, numerous DNA repair pathways that specialize in repair of specific types of DNA damage have evolved. Nucleotide excision repair (NER) serves to repair DNA lesions induced by UV light or by chemical modification of DNA bases. Because NER is critical for cellular function, it is important for human health. Mutations affecting the molecular components of the pathway cause human diseases, including xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Additionally, NER is able to remove DNA lesions caused by cancer chemotherapeutics and may reduce efficacy of treatment. A mechanistic understanding of NER, its regulation, and disease-causing mechanisms is therefore critical. This requires structural information on the complex molecular assemblies that perform the repair reaction.

I aim to elucidate the structural mechanism of NER by using cryo-electron microscopy to determine the structures of repair intermediates along the NER pathway. Additionally, I aim to perform structural studies of disease mutations that affect the crucial NER factor TFIIH. To this end, I am reconstituting the pathway from recombinantly expressed and purified components, which can be obtained in larger amounts than endogenously purified material and can harbour engineered mutations if required. The cryo-electron microscopy experiments will be complemented by structure-guided functional investigation of NER in a collaboration with the laboratory of Prof. Wojciech Niedzwiedz. The combined structural and functional studies will enable a mechanistic understanding of the function and dysfunction of NER.