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

维持正确的遗传信息对所有生物都是至关重要的。突变是遗传疾病和癌症的主要原因，也可能与衰老有关。80%到90%的人类癌症最终都是由于DNA损伤。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）是一种普遍存在于所有生物体内的DNA修复机制。它是独特的在它的多功能性修复广泛的病变。在人类中，NER是保护DNA免受紫外线损伤的主要修复机制。参与NER的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、科凯恩综合征和毛硫营养不良。通过结合生化和结构研究，我们将分析UvrA-UvrB和XPD蛋白识别损伤的过程。我们将通过冷冻电子显微镜和晶体学方法的结合来追求不同的原核生物UvrA-，UvrB-和UvrA-UvrB- dna复合物。XPD的结构研究将在其核苷酸辅因子的存在下以及与DNA的复合物中进行。通过位点定向诱变、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点。基于我们之前对原核生物UvrABC系统的研究，我们将在受损DNA存在的情况下对XPD进行表征，以了解导致预切口复合物的必要步骤，这是修复过程中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.