Regulation of Nucleotide Excision Repair by Proteolysis

描述（由申请人提供）：核苷酸切除修复（NER）通过去除紫外线照射或化学诱变剂引起的螺旋扭曲DNA损伤，在维持基因组完整性方面起着重要作用。NER中的缺陷是人类遗传性疾病着色性干皮病的基础，其特征是对紫外线敏感和皮肤癌的高发病率。对NER的研究主要集中在确定切除和修复反应的组分和生物化学上，而对细胞控制NER活性的调节机制知之甚少。Cullin 4A泛素化机制最近被证明可以介导受损DNA结合蛋白的p48亚基的泛素依赖性蛋白水解，这被认为参与了NER的初始DNA损伤识别步骤。我们的长期目标是了解泛素蛋白水解途径如何调节NER，并将这种理解与与NER缺陷相关的人类疾病以及泛素化机制的故障联系起来。本应用的目的是了解cull - 4a介导的p48降解在控制核苷酸切除修复的损伤传感步骤中的分子基础、调控途径和功能意义。该应用的中心假设是CUL-4A泛素化机制通过限制p48的丰度来控制NER机制识别和去除特定DNA损伤的能力。提出的具体目标是：(1)确定CUL- 4A/DDB相互作用和DDB蛋白亚细胞分布的分子基础。(2)确定CUL-4A在DNA损伤识别和修复中的功能意义。(3)评估c-Abl在调控p48降解和核苷酸切除修复中的作用。本研究结果将为泛素依赖性蛋白水解调控核苷酸切除修复提供新的范式，并对控制DDB蛋白胞内分布和丰度的生化机制有更好的理解。这些研究的完成也将揭示CUL- 4A的异常激活如何促进肿瘤的发展。

DESCRIPTION (provided by applicant): Nucleotide excision repair (NER) plays an important role in maintaining genomic integrity through removing helix-distorting DNA damages caused by UV irradiation or chemical mutagens. Defects in NER underlie the human hereditary disease, xeroderma pigmentosum, which is characterized as sensitivity to ultraviolet light and a high incidence of skin cancer. Studies of NER have been focused on identifying components and the biochemistry of excision and repair reactions, while little is known about the regulatory mechanisms cells employ to control the NER activity. The Cullin 4A ubiquitination machinery has recently been shown to mediate ubiquitin-dependent proteolysis of the p48 subunit of damaged DNA binding proteins that are believed to participate in the initial DNA damage recognition step of NER. Our long-range goal is to understand how the ubiquitin-proteolytic pathway regulates NER, and to relate this understanding to human diseases associated with defective NER as well as the malfunctions of the ubiquitination machinery. The objective of this application is to understand the molecular basis, the regulatory pathways, and the functional significance of CUL-4A-mediated p48 degradation in controlling the damage-sensing step of nucleotide excision repair. The central hypothesis of the application is that the CUL-4A ubiquitination machinery controls the ability of the NER machinery to recognize and remove specific DNA damages through restricting the abundance of p48. The specific aims proposed are (1) To determine the molecular basis for CUL- 4A/DDB interactions and for subcellular distribution of DDB proteins. (2) To determine the functional significance of CUL-4A in DNA damage recognition and repair. (3) To assess the role of c-Abl in regulating p48 degradation and nucleotide excision repair. The results of this work will provide a new paradigm for the regulation of nucleotide excision repair by ubiquitin-dependent proteolysis, and generate a better understanding of the biochemical mechanisms controlling the intracellular distribution and abundance of DDB proteins. Completion of the proposed studies will also shed light on how abnormal activation of CUL- 4A contributes to tumor development.