Regulation of Nucleotide Excision Repair by Proteolysis

描述(申请人提供)：核苷酸切除修复(NER)通过去除紫外线辐射或化学诱变剂引起的扭曲螺旋的DNA损伤，在维持基因组完整性方面发挥着重要作用。NER缺陷是人类遗传性疾病着色性干皮病的基础，该疾病的特点是对紫外线敏感，皮肤癌的发病率很高。对NER的研究主要集中在识别NER的成分以及切除和修复反应的生化反应上，而对细胞用来控制NER活性的调控机制知之甚少。最近的研究表明，Cullin 4A泛素化机制可以介导受损DNA结合蛋白P48亚单位的泛素依赖的蛋白分解，这些结合蛋白被认为参与了NER的初始DNA损伤识别步骤。我们的长期目标是了解泛素-蛋白分解途径如何调节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CuL-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机制识别和移除特定DNA损伤的能力。提出的具体目标是(1)确定CuL-4A/DDB相互作用的分子基础和DDB蛋白的亚细胞分布。(2)确定CuL-4A在DNA损伤识别和修复中的功能意义。(3)探讨c-Abl在调节P48降解和核苷酸切除修复中的作用。这项工作的结果将为泛素依赖的蛋白分解调节核苷酸切除修复提供一个新的范式，并使人们更好地理解控制DDB蛋白在细胞内分布和丰度的生化机制。拟议研究的完成还将阐明CUL-4A的异常激活如何有助于肿瘤的发展。

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.