Regulation of Nucleotide Excision Repair by Proteolysis

蛋白水解调节核苷酸切除修复

• 批准号: 8245783
• 负责人: Pengbo Zhou
• 金额: $ 32.06万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8245783
• 关键词: ABL1 gene; Address; Affect; Animal Model; Animals; BRCA1 gene; Binding; Binding Proteins; Biochemical; Biochemical Genetics; Biochemical Reaction; Biological; Biological Assay; CDKN1A gene; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Survival; Cells; Chemicals; Complex; DNA; DNA Damage; DNA Repair; DNA repair protein; DNA-Binding Proteins; Development; Disease; Ensure; Event; Excision; Excision Repair; Funding; Genomic Instability; Genomics; Goals; Hand; Health; Hour; Human; Kinetics; Knockout Mice; Knowledge; Malignant Neoplasms; Mediating; Modification; Molecular Genetics; Mus; Mutagens; Mutate; Normal Cell; Nucleotide Excision Repair; Pathway interactions; Personal Satisfaction; Phosphotransferases; Physiological; Play; Positioning Attribute; Prevention; Prevention strategy; Proteins; Proteolysis; Reagent; Regulation; Regulatory Pathway; Resistance; Role; Site; Skin; Skin Cancer; Skin Carcinogenesis; Skin Neoplasms; Structure; Testing; Time; Tumor Suppression; Tumorigenicity; Ubiquitin; Ubiquitination; Ultraviolet Rays; Up-Regulation; Xeroderma Pigmentosum; base; c-abl Proto-Oncogenes; cancer cell; carcinogenesis; cell growth; cullin 4A; design; histone modification; improved; in vivo; insight; irradiation; keratinocyte; multicatalytic endopeptidase complex; mutant; novel; novel strategies; oncoprotein p21; photolesion; prevent; public health relevance; repaired; sensor; structural biology; tumor; tumorigenesis; ubiquitin ligase

DESCRIPTION (provided by applicant): Nucleotide excision repair (NER) is the major DNA repair machinery that removes DNA damage induced by ultraviolet light (UV) and chemical mutagens to prevent genomic instability and tumorigenesis. While the enzymatic reactions for excision and repair of DNA photolesions are well studied, regulatory pathways governing the temporal and spatial control of DNA damage recognition remains poorly understood, and the physiological functions of such regulation on tumor suppression have not been explored due to the unavailability of animal models. The cullin 4A (CUL-4A) ubiquitin ligase has recently emerged as a key regulator of two DNA damage sensors: the damaged DNA binding proteins (DDBs, heterodimers of DDB1 and DDB2) and xeroderma pigmentosum complementation group C (XPC) protein. Interestingly, recent studies revealed a second function of DDBs as integral components of the CUL-4A ubiquitin ligase complex. During the previous funding period, our biochemical and structural biology studies provided mechanistic insight into the assembly of the CUL-4A-DDB complex, and a novel kinase-independent function of c-Abl in activating CUL-4A-dependent ubiquitination of DDBs both under normal conditions and upon UV irradiation. Importantly, we generated conditional CUL-4A knockout mice and showed that skin-specific CUL-4A knockout mice were resistant to UV-induced skin carcinogenesis, suggesting an intriguing possibility of pharmacological inhibition of CUL-4A as a prevention strategy for UV-induced skin cancer. While CUL-4B shares overlapping functions with CUL-4A in cell growth and survival, its role on DDB2 degradation and NER appears less pronounced than that of CUL-4A. We also collaborated with Dr. Stephan Goff to determine the physiological functions of DDB1 in NER and in controlling cell cycle and genomic integrity in the conditional DDB1 knockout mice. Interestingly, our in vivo studies revealed dramatic upregulation of the cyclin-dependent kinase inhibitor p21/CIP1/WAF1 in CUL-4A-/- and DDB1-/- mice, as well as in MEF cells and keratinocytes derived from these mice. Our long-term goal is to understand how the ubiquitin pathway regulates DNA repair and affects tumor development. We hypothesize that the CUL-4A and CUL-4B ubiquitin ligase activity is precisely controlled both to ensure proper execution of NER and to halt cell cycle events to allow time for efficient repair. We are uniquely positioned to test this hypothesis since we have generated specific ubiquitination-resistant DDB2 mutants, identified a novel modulator (BRAP2) in the temporal control of CUL-4A activity following UV irradiation, and have CUL-4A, DDB1 and p21 (or CIP1 or WAF1) knockout mice in hand. We propose to employ a combination of biochemical, genetic and cell biological approaches to address the following three specific aims: (1) establish the mechanism by which DDB2 ubiquitination regulates damage recognition and repair; (2) To determine the temporal control of CUL-4A and CUL-4B ubiquitin ligase activity by BRAP2 during NER; (3) To determine the mechanistic basis and functional significance of p21 accumulation in protecting CUL-4A-deficient mice against UV-induced carcinogenesis. Successful completion of these aims will significantly contribute to our understanding of the molecular and genetic basis of the ubiquitin-proteasome pathway in DNA repair and tumorigenesis. Knowledge gained from these efforts could be exploited to devise novel strategies for the prevention and/or treatment of UV- and chemical mutagen-induced skin cancer or skin-related disorders, and thus improve the health and well-being of humans.

描述（由申请人提供）：核苷酸切除修复（NER）是主要的DNA修复机制，可消除紫外线（UV）和化学诱变剂诱导的DNA损伤，以防止基因组不稳定和肿瘤发生。虽然DNA光损伤切除和修复的酶促反应已得到充分研究，但对DNA损伤识别的时间和空间控制的调控途径仍知之甚少，并且由于缺乏动物模型，这种调控对肿瘤抑制的生理功能尚未得到探索。 cullin 4A (CUL-4A) 泛素连接酶最近已成为两种 DNA 损伤传感器的关键调节因子：受损的 DNA 结合蛋白（DDB、DDB1 和 DDB2 的异二聚体）和着色性干皮病互补组 C (XPC) 蛋白。有趣的是，最近的研究揭示了 DDB 作为 CUL-4A 泛素连接酶复合物的组成部分的第二个功能。在之前的资助期间，我们的生化和结构生物学研究提供了对 CUL-4A-DDB 复合物组装的机制见解，以及 c-Abl 在正常条件下和紫外线照射下激活 CUL-4A 依赖性 DDB 泛素化的新型激酶独立功能。重要的是，我们生成了条件性 CUL-4A 基因敲除小鼠，并表明皮肤特异性 CUL-4A 基因敲除小鼠对紫外线诱导的皮肤癌具有抵抗力，这表明药物抑制 CUL-4A 作为紫外线诱导的皮肤癌的预防策略的有趣可能性。虽然 CUL-4B 在细胞生长和存活方面与 CUL-4A 具有重叠的功能，但其对 DDB2 降解和 NER 的作用似乎不如 CUL-4A 明显。我们还与 Stephan Goff 博士合作，确定了 DDB1 在 NER 中的生理功能以及在条件 DDB1 敲除小鼠中控制细胞周期和基因组完整性的功能。有趣的是，我们的体内研究揭示了 CUL-4A-/- 和 DDB1-/- 小鼠以及源自这些小鼠的 MEF 细胞和角质形成细胞中细胞周期蛋白依赖性激酶抑制剂 p21/CIP1/WAF1 的显着上调。我们的长期目标是了解泛素通路如何调节 DNA 修复并影响肿瘤的发展。我们假设 CUL-4A 和 CUL-4B 泛素连接酶活性受到精确控制，以确保 NER 的正确执行并停止细胞周期事件，以便有时间进行有效修复。我们具有独特的优势来检验这一假设，因为我们已经产生了特定的泛素化抗性 DDB2 突变体，鉴定了一种新型调节剂 (BRAP2)，可在紫外线照射后控制 CUL-4A 活性，并拥有 CUL-4A、DDB1 和 p21（或 CIP1 或 WAF1）敲除小鼠。我们建议采用生化、遗传和细胞生物学相结合的方法来解决以下三个具体目标：（1）建立DDB2泛素化调节损伤识别和修复的机制； (2)确定NER过程中BRAP2对CUL-4A和CUL-4B泛素连接酶活性的时间控制； (3) 确定p21积累在保护CUL-4A缺陷小鼠免受紫外线诱导的致癌作用中的机制基础和功能意义。成功完成这些目标将极大地有助于我们了解 DNA 修复和肿瘤发生中泛素-蛋白酶体途径的分子和遗传基础。从这些努力中获得的知识可用于设计预防和/或治疗紫外线和化学诱变剂诱发的皮肤癌或皮肤相关疾病的新策略，从而改善人类的健康和福祉。