Molecular interplay in eukaryotic nucleotide excision repair

许多内源性和外源性因子不断地破坏我们的DNA。据估计，一个人体细胞每天会发生多少次损伤，从10到4到10到6。假设一个成年人的身体含有10的12次方个细胞，那么修复机器每天必须修复10的16次方到10的18次方个损伤。因此，80%至90%的人类癌症最终是由于DNA损伤，这并不奇怪。在细胞可用的各种DNA修复机制中，核苷酸切除修复（NER）是最有趣的途径，就其广泛的底物特异性而言。它是一种普遍的DNA修复机制，存在于所有三个生命王国中，并以其去除大体积DNA损伤的能力而闻名。在人类中，NER是保护DNA免受紫外线损伤的唯一修复机制。NER相关缺陷基因的表型后果在三种严重疾病中是明显的：着色性干皮病、Cockaynes综合征和甲状腺营养不良。这条途径的多功能性和它的严重后果，当它失败时，要求的方法来破译的能力，识别和修复基板，其差异如此巨大的大小和组成，并获得一个全面的了解这条pathway.The十个亚基含有转录因子TFIIH核苷酸切除修复中起着核心作用和TFIIH内的解旋酶XPD是必不可少的损伤验证过程。通过结合生物化学，生物物理和结构研究，我们将分析TFIIH内不同亚基之间的相互作用，特别关注XPD及其相互作用伙伴。我们将继续对真核XPD及其与直接调节因子p44和MAT 1亚基形成的复合物进行结构和功能研究。XPD的结构研究将在ATP和ATP类似物以及不同的DNA底物存在下进行。为了破译XPD的次级相互作用伴侣的调节作用，我们还将确定p44-p34复合物的结构，该复合物将XPD连接到TFIIH的核心。该结构将揭示XPD-p44复合物如何通过TFIIH核心内的蛋白质相互作用锚定和调节。我们的结构工作将伴随着XPD和拟议的复合物的详细的生化表征。这种组合方法将为调节XPD的分子网络和真核NER中的损伤验证步骤提供前所未有的见解。

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.