The functional and molecular architecture of core TFIIH

• Applicant: Professorin Dr. Caroline Kisker
• Subject Area: Structural Biology
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 383268694

Responses to DNA damage are essential for the maintenance of genome integrity. Nucleotide excision repair (NER) corrects DNA-damages resulting from UV irradiation or drugs such as cis-platinum derivatives. Mutations in NER genes lead to severe human diseases such as Xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or a combination of XP and Cockayne syndrome (XP/CS). The general transcription factor II H (TFIIH) plays a central role in both transcription and NER. In its entirety TFIIH consists of a total of 10 subunits of which XPB, p62, p52, p44, p34, and p8 build the core. The CDK7, MAT1, and cyclin H subunits constitute the CAK, a kinase complex involved in promoter escape that also participates in transcription regulation. Core-TFIIH and CAK are bridged by XPD which is also present in other non-TFIIH complexes. Our studies showed that all core TFIIH components especially the two helicases XPD and XPB from the eukaryotic model organism Chaetomium thermophilum can be produced in sufficient quantity and quality for biochemical and structural analyses. This system thus provides the unique opportunity to analyze the individual contributions of each subunit to the overall function of core TFIIH. In our proof of principle analyses we showed that data obtained for XPD from C. thermophilum can be directly correlated with human XPD. We will further exploit this strategy using the C. thermophilum proteins to decipher the role of the other subunits of TFIIH with an emphasis on the two helicases XPB and XPD and their regulatory subunits during NER and transcription initiation. In order to obtain a complete molecular view of core TFIIH, our biochemical data will be accompanied by a detailed structural characterization using single particle cryo electron microscopy . No high resolution structural data with respect to core TFIIH are available so far: How do the core TFIIH subunits interact with each other, how does core TFIIH regulate its XPB and XPD helicase/ATPase activities during NER or transcription are, among many others, open key questions. We will dissect the different functions within TFIIH using a combination of the aforementioned techniques. Building up on initial protein-protein combinations that regulate XPD or XPB, which will be analyzed by activity and interaction studies, we will resolve the building blocks that reconstitute active core TFIIH. Subsequently, we will generate active core TFIIH complexes that will be amenable for a detailed biochemical characterization and structural analysis using single particle cryo electron microscopy, yielding unrivaled molecular insights into the events that lead to the successful assembly of a core TFIIH complex.

• DFG Programme: Research Grants