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，可清除紫外线（UV）和化学诱变剂诱导的DNA损伤，以防止基因组不稳定性和肿瘤发生。虽然切除和修复的DNA光损伤的酶促反应进行了很好的研究，管理的时间和空间控制的DNA损伤识别的调节途径仍然知之甚少，和肿瘤抑制的生理功能，这种调节尚未探索由于动物模型的不可用。cullin 4A（CUL-4A）遍在蛋白连接酶最近成为两种DNA损伤传感器的关键调节因子：受损的DNA结合蛋白（DDB，DDB 1和DDB 2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DDBs作为CUL-4A泛素连接酶复合物的组成部分的第二种功能。在之前的资助期间，我们的生物化学和结构生物学研究提供了对CUL-4A-DDB复合物组装的机制性见解，以及c-Abl在正常条件下和UV照射下激活CUL-4A依赖的DDB泛素化的新型激酶独立功能。重要的是，我们产生了条件性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UV诱导的皮肤癌发生具有抗性，这表明了CUL-4A的药理学抑制作为UV诱导的皮肤癌预防策略的有趣可能性。虽然CUL-4 B在细胞生长和存活中与CUL-4A具有重叠的功能，但其对DDB 2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DDB 1在NER中的生理功能，以及在条件DDB 1敲除小鼠中控制细胞周期和基因组完整性的生理功能。有趣的是，我们的体内研究揭示了细胞周期蛋白依赖性激酶抑制剂p21/CIP 1/WAF 1在CUL-4A-/-和DDB 1-/-小鼠以及来自这些小鼠的MEF细胞和角质形成细胞中的显著上调。我们的长期目标是了解泛素途径如何调节DNA修复和影响肿瘤的发展。我们假设CUL-4A和CUL-4 B泛素连接酶活性受到精确控制，以确保正确执行NER并停止细胞周期事件，以便有时间进行有效修复。我们是唯一的定位来测试这一假设，因为我们已经产生了特定的泛素化抗性DDB 2突变体，确定了一种新的调制器（BRAP 2）的时间控制CUL-4A活动后，紫外线照射，并有CUL-4A，DDB 1和p21（或CIP 1或WAF 1）基因敲除小鼠在手。本研究拟采用生物化学、遗传学和细胞生物学相结合的方法，研究以下三个方面的具体目标：（1）建立DDB 2泛素化调控损伤识别和修复的机制;（2）确定BRAP 2在NER过程中对CUL-4A和CUL-4 B泛素连接酶活性的时间调控;（3）探讨p21蛋白在CUL-4A基因缺陷小鼠体内的积累对紫外线致癌的保护作用及其机制。这些目标的成功完成将大大有助于我们理解DNA修复和肿瘤发生中的泛素-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导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。