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，可去除由紫外线（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。虽然对切除和修复DNA光子的酶促反应进行了充分的研究，但管理DNA损伤识别的时间和空间控制的调节途径仍然很了解，并且由于动物模型的不可用而没有探索这种调节对肿瘤抑制的生理功能。 Cullin 4A（CUL-4A）泛素连接酶最近成为两个DNA损伤传感器的关键调节剂：受损的DNA结合蛋白（DDBS，DDB1和DDB2的异二聚体）和Xeroderma sigmentosum copmentosum copmentosum copmentosum copmentation copmentation contemume contement corpectimum cornectation compection cormentation compection c（xpc）蛋白质。有趣的是，最近的研究揭示了DDB作为CUL-4A泛素连接酶复合酶复合物的积分成分的第二个功能。在上一个资金期间，我们的生化和结构生物学研究为CUL-4A-DDB复合物的组装提供了机械洞察力，以及C-ABL在正常条件和UV辐射下激活CUL-4A依赖性DDB的新型激酶独立的功能。重要的是，我们产生了有条件的CUL-4A基因敲除小鼠，并表明皮肤特异性的CUL-4A基因敲除小鼠对紫外线诱导的皮肤致癌作用有抵抗力，这表明对CUL-4A的药理抑制作用引起了人们对紫外线诱发的皮肤癌的预防策略的有趣可能性。尽管CUL-4B在细胞生长和存活中具有与CUL-4A的重叠函数，但其​​在DDB2降解上的作用和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NER中DDB1的生理功能，并控制条件DDB1敲除小鼠中的细胞周期和基因组完整性。有趣的是，我们的体内研究表明，在CUL-4A - / - 和DDB1 - / - 小鼠以及MEF细胞和角质类细胞中，细胞周期蛋白依赖性激酶抑制剂p21/CIP1/WAF1的急剧上调。我们的长期目标是了解泛素途径如何调节DNA修复并影响肿瘤的发育。我们假设CUL-4A和CUL-4B泛素连接酶的活性得到了精确控制，以确保正确执行NER并停止细胞周期事件，以使时间有效修复。由于我们已经在紫外线照射后的CUL-4A活性的时间控制中确定了一种新型的调节剂（BRAP2），因此我们可以独特地测试这一假设，因为我们已经生成了特定的泛素化DDB2突变体，并具有CUL-4A，DDB1和P21（或P21（或CIP1或WAF1）的静止元素。我们建议采用生化，遗传和细胞生物学方法的组合来解决以下三个特定目的：（1）确定DDB2泛素化调节损害识别和修复的机制； （2）确定NER期间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方面的机理基础和功能意义。这些目标的成功完成将显着有助于我们理解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。可以利用从这些努力中获得的知识来制定预防和/或治疗紫外线和化学诱变引起的皮肤癌或与皮肤相关疾病的新型策略，从而改善了人类的健康和福祉。