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

保持正确的遗传信息对所有生物来说都是至关重要的。突变是遗传性疾病和癌症的主要原因，也可能与衰老有关。80%到90%的人类癌症最终是由DNA损伤引起的。因此，进化出了不同的修复机制来保护基因组。核苷酸切除修复(NER)是一种在所有生物中普遍存在的主要DNA修复机制。它的独特之处在于它的多功能性，可以修复广泛的损伤。在人类中，NER是保护DNA免受紫外线损伤的主要修复机制。NER基因缺陷在色素性干皮病、Cockayne综合征和毛发硫代营养不良三种严重疾病中的表型后果是明显的。通过生化和结构研究相结合的方法，我们将分析UvrA-UvrB和XPD蛋白的损伤识别过程。我们将通过低温电子显微镜和结晶学方法相结合的方法来探索不同的原核生物UvrA-、UvrB-和UvrA-UvrB-DNA复合体。XPD的结构研究将在其核苷酸辅因子存在的情况下进行，以及在与DNA的复合体中进行。通过定点突变、生化和生物物理研究，我们将确定DNA结合、双链分离和损伤验证的位置。在我们以前对原核生物UvrABC系统的研究的基础上，我们将在DNA受损的情况下对XPD进行表征，以了解导致切前复合体的必要步骤，这是修复过程的所有后续反应的先决条件。

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.