Molecular interplay in eukaryotic nucleotide excision repair

• Applicant: Professorin Dr. Caroline Kisker
• Subject Area: Biochemistry
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269187088

Numerous endogenous and exogenous agents constantly damage our DNA. Estimates of how many damages occur within a single human cell range from 10 to the 4th to 10 to the 6th per day. Assuming that an adult human body contains 10 to the 12th cells, the repair machinery thus has to repair 10 to the 16th to 10 to the 18th damages per day. It is thus not surprising that 80 to 90% of all human cancers are ultimately due to DNA damageAmong the various DNA repair mechanisms available to the cell, nucleotide excision repair (NER) is the most intriguing pathway with respect to its broad substrate specificity. It is a universal DNA repair mechanism found in all three kingdoms of life and is well known for its ability to remove bulky DNA lesions. In humans, NER is the only 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, Cockaynes syndrome and trichothiodystrophy. The versatility of this pathway and its serious consequences when it fails calls for approaches to decipher the ability to recognize and repair substrates, which differ so vastly in size and composition and to gain an overall understanding of this pathway.The ten subunit containing transcription factor TFIIH plays a central role in nucleotide excision repair and the helicase XPD within TFIIH is essential for the damage verification process. Through a combination of biochemical, biophysical and structural studies we will analyze the interplay between the different subunits within TFIIH with a special focus on XPD and its interaction partners. We will pursue structural and functional studies on a eukaryotic XPD and the complexes it forms with its direct regulators, the p44 and MAT1 subunits. Structural studies on XPD will be pursued in the presence of ATP and ATP-analogues as well as with different DNA substrates. To decipher the regulatory role of secondary interaction partners of XPD we will also determine the structure of the p44-p34 complex, which bridges XPD to the core of TFIIH. This structure will reveal how the XPD-p44 complex is anchored and regulated via protein interactions within the TFIIH-core. Our structural efforts will be accompanied by a detailed biochemical characterization of XPD and the proposed complexes. This combinatorial approach will provide unprecedented insight into the molecular network regulating XPD and the damage verification step in eukaryotic NER.

• DFG Programme: Research Grants