The functional and molecular architecture of core TFIIH

对DNA损伤的反应对于维持基因组的完整性是必不可少的。核苷酸切除修复(NER)纠正紫外线辐射或药物(如顺铂衍生物)造成的DNA损伤。NER基因突变会导致严重的人类疾病，如着色性干皮病(XP)、毛发硫代营养不良(TTD)或XP和Cockayne综合征(XP/CS)的组合。通用转录因子II H(TFIIH)在转录和NER中都起着核心作用。TFIIH总共由10个亚基组成，其中xpb、p62、p52、p44、p34和p8构成核心。CDK7、MAT1和Cyclin H亚基组成了CAK，这是一种参与启动子逃逸的激酶复合体，也参与转录调控。CORE-TFIIH和CAK通过XPD桥联，XPD也存在于其他非TFIIH复合体中。我们的研究表明，TFIIH的所有核心成分，特别是真核模式生物嗜热毛壳菌的两个解旋酶XPD和XPB，都能产生足够数量和质量的解旋酶，用于生化和结构分析。因此，这一系统提供了独特的机会来分析每个亚单位对核心TFIIH整体功能的单独贡献。在我们的原理证明分析中，我们证明了从嗜热梭菌获得的XPD数据可以直接与人类XPD相关。我们将使用嗜热链霉菌蛋白进一步利用这一策略来破译TFIIH的其他亚基的作用，重点是两个解旋酶XPB和XPD及其在NER和转录启动过程中的调节亚基。为了获得核心TFIIH的完整分子视图，我们的生化数据将伴随着使用单粒子低温电子显微镜的详细结构表征。到目前为止，还没有关于核心TFIIH的高分辨率结构数据：核心TFIIH亚基如何相互作用，核心TFIIH如何在NER或转录过程中调节其XPB和XPD解旋酶/ATPase活性，这些都是许多开放的关键问题。我们将结合使用上述技术来分析TFIIH中的不同功能。在调节XPD或XPB的初始蛋白质-蛋白质组合的基础上，我们将解决重建活性核心TFIIH的构建块，这将通过活性和相互作用研究进行分析。随后，我们将生成活性的核心TFIIH复合体，这些复合体将能够使用单颗粒低温电子显微镜进行详细的生化表征和结构分析，从而对导致核心TFIIH复合体成功组装的事件产生无与伦比的分子洞察力。

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.