Coordination of the late steps of human nucleotide excision repair

描述(申请人提供)：核苷酸切除修复(NER)是一种多功能的DNA修复途径，涉及从DNA中移除不同的损伤，包括那些由紫外线形成的损伤。易患癌症的遗传性色素性干皮病是由多种NER基因缺陷引起的，这一事实突显了NER的重要性。NER通过30多种蛋白质的协同作用发挥作用，这些蛋白质依次在紫外线损伤的部位组装。尽管NER涉及的基本生化交易已经被识别，但人们对个体步骤是如何协调以确保顺利通过该途径的了解较少。本研究结合生化、细胞生物学、化学和结构学方法，研究NER中双重切开和修复合成步骤的调节和协调。该建议是以这样的假设为指导的，即ERCC1-XPF到病变的5‘和XPG到病变的3’的切口是暂时和空间调节的，以避免在这个过程中形成有害的断裂和间隙。我们将检验这种协调是通过蛋白质-蛋白质相互作用、DNA结合和催化活性实现的具体假设如下：1)新的结构信息将被用于重新分配和表征XPA-ERCC1相互作用结构域，我们将研究XPA如何将ERCC1-XPF招募到NER位点。2)我们将研究ERCC1-XPF的单个DNA结合域如何通过NER途径对其催化活性和进展做出贡献。3)我们将研究ERCC1-XPF和XPG的切割顺序以及切割和修复合成活动的协调如何避免在切除含有损伤的30个核苷酸长的寡核苷酸后形成单链DNA缺口。我们的研究将为人类细胞中复杂生化途径的调控提供新的分子基础，以及遗传性疾病和癌症的分子基础。由于NER蛋白还可以中和许多临床上重要的抗肿瘤药物的作用，我们的研究也将为治疗干预提供新的靶点。 项目简介：核苷酸切除修复(NER)是一种复杂的DNA修复途径，可以抵消紫外线、环境制剂和癌症化疗药物对DNA的损伤。NER基因的缺陷导致了易患癌症的遗传性疾病着色性干皮病，我们对这一途径的跨学科研究有助于我们理解人类疾病的分子基础。由于NER蛋白也参与了肿瘤细胞对抗癌药物的耐药性，我们的研究也可能为抗肿瘤治疗提供新的靶点。

DESCRIPTION (provided by applicant): Nucleotide Excision Repair (NER) is a versatile DNA repair pathway involved in the removal of diverse lesions, including those formed by UV light, from DNA. The importance of NER is underscored by the fact that the cancer-prone inherited disorder xeroderma pigmentosum is caused by defects in various NER genes. NER acts by the concerted action of over 30 proteins that sequentially assemble at sites of UV damage. Although the basic biochemical transactions involved in NER have been discerned, it is less well understood how the individual steps are coordinated to ensure smooth progression through the pathway. The research outlined here combines biochemical, cell biological, chemical and structural approaches to study the regulation and coordination of the dual incision and repair synthesis steps in NER. The proposal is guided by the hypothesis that the incisions, 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG, are temporarily and spatially regulated to avoid the formation of deleterious breaks and gaps in the process. We will test the specific hypothesis that this coordination is achieved by protein-protein interactions, DNA binding and catalytic activities as follows: 1) New structural information will be used to reassign and characterize the XPA-ERCC1 interaction domains and we will study how XPA recruits ERCC1-XPF to sites of NER. 2) We will study how individual DNA binding domains of ERCC1-XPF contribute to its catalytic activity and progression through the NER pathway. 3) We will investigate how the order of incisions by ERCC1-XPF and XPG and the coordination of incision and repair synthesis activities avoids the formation of single-stranded DNA gaps following excision of a 30 nucleotide long oligonuelcotide containing the damage. Our studies will provide new insight into the molecular basis of the regulation of a complex biochemical pathway in human cells as well as the molecular basis of inherited disease and the etiology of cancer. Since NER proteins also counteract the action of many clinically important antitumor agents, our studies will also contribute new targets for therapeutic intervention. Project Narrative: Nucleotide excision repair (NER) is a complex DNA repair pathway that counteracts damage to DNA caused by UV light, environmental agents and cancer chemotherapeutic drugs. Defects in NER genes result in the cancer-prone inherited disorder xeroderma pigmentosum and our interdisciplinary studies of this pathway contribute to our understanding of the molecular basis of human disease. Since NER proteins also contribute to the resistance of tumor cells to anti-cancer agents our investigations may also provide new targets for anti- tumor therapy.