The structural and functional characterization of the XPD and UvrA-UvrB proteins involved in Nucleotide Excision Repair

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Maintenance of the correct genetic information is crucial for all living organisms. Mutations are the primary cause of hereditary diseases, as well as cancer, and may also be involved in aging. 80 to 90% of all human cancers are ultimately due to DNA damage. Consequently, different repair mechanisms have evolved to protect the genome. Nucleotide excision repair (NER) is a major DNA repair mechanism that is universal among all biological organisms. It is unique in its versatility to repair a broad range of lesions. In humans, NER is the major repair mechanism to protect DNA from damage induced by ultraviolet light. The phenotypic consequences of defective genes involved in NER are apparent in three severe diseases: xeroderma pigmentosum, Cockayne’s syndrome and trichothiodystrophy.Through a combination of biochemical and structural studies we will analyze the process of damage recognition by the UvrA-UvrB and XPD proteins. We will pursue the different prokaryotic UvrA-,UvrB-, and UvrA-UvrB-DNA complexes by a combination of cryo electron microscopy and crystallographic methods. Structural studies of XPD will be pursued in the presence of its nucleotide cofactor as well as in complex with DNA. Through site directed mutagenesis, biochemical and biophysical studies we will define the sites for DNA binding, double strand separation and damage verification. Based on our previous studies on the prokaryotic UvrABC system we will characterize XPD in the presence of damaged DNA to obtain an understanding of the necessary steps leading to the pre-incision complex, which is a pre-requisite for all subsequent reactions of the repair process.