Regulation of Nucleotide Excision Repair by Proteolysis

描述(申请人提供)：核苷酸切除修复(NER)是主要的DNA修复机制，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。虽然对DNA光渗漏的切除和修复的酶反应已经有了很好的研究，但控制DNA损伤识别的时间和空间调控的调控途径仍然知之甚少，而且由于缺乏动物模型，这种调控在肿瘤抑制中的生理功能还没有被探索。最近，cullin 4A(CuL-4A)泛素连接酶被认为是两个DNA损伤感受器的关键调节因子：受损的DNA结合蛋白(DDBs，DDB1和DDB2的异源二聚体)和着色性干皮病互补C组蛋白(XPC)。有趣的是，最近的研究揭示了DDB的第二个功能，即作为CuL-4A泛素连接酶复合体的组成部分。在之前的资助期间，我们的生化和结构生物学研究为CuL-4A-DDB复合体的组装提供了机械性的见解，并提供了c-Abl在正常条件下和紫外线照射下激活依赖于CuL-4A的DDB泛素化的新的不依赖于激酶的功能。重要的是，我们获得了条件性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癌具有抵抗力，这表明CUL-4A作为紫外线诱导的皮肤癌的预防策略具有耐人寻味的可能性。虽然CuL-4B在细胞生长和存活方面与CuL-4A有重叠的功能，但它在DDB2NER降解和NER方面的作用似乎没有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，以及在条件性DDB1基因敲除小鼠中控制细胞周期和基因组完整性的作用。有趣的是，我们的体内研究显示，周期蛋白依赖的激酶抑制物p21/CIP1/WAF1在CUL-4A-/-和DDB1-/-小鼠以及从这些小鼠衍生的MEF细胞和角质形成细胞中显著上调。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。我们推测，CuL-4A和CUL-4B泛素连接酶的活性受到精确控制，以确保NER的正确执行，并阻止细胞周期事件，以便有时间进行有效的修复。我们处于独特的地位来检验这一假设，因为我们已经产生了特定的泛素化抗性DDB2突变体，确定了一种新的调节剂(BRAP2)在紫外线照射后对CuL-4A活性进行时间控制，并拥有CuL-4A、DDB1和p21(或CIP1或WAF1)基因敲除小鼠。我们建议结合生化、遗传学和细胞生物学的方法来解决以下三个特定目标：(1)建立DDB2泛素化调控损伤识别和修复的机制；(2)确定BRAP2在NER过程中对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中的机制基础和功能意义。这些目标的成功实现将有助于我们理解泛素-蛋白酶体途径在DNA修复和肿瘤发生中的分子和遗传学基础。从这些努力中获得的知识可以被用来制定新的战略，以预防和/或治疗紫外线和化学诱变剂引起的皮肤癌或与皮肤相关的疾病，从而改善人类的健康和福祉。与公共卫生相关：DNA损伤导致80%-90%的人类癌症的发生。泛素依赖的DNA修复蛋白的蛋白分解对于调节正常细胞和癌细胞的DNA损伤识别和去除功能至关重要。这项研究将表征cullin 4A泛素连接酶在控制核苷酸切除修复装置的损伤传感器稳定性方面的作用。从这些努力中获得的知识可以被用来制定新的战略，以预防和/或治疗紫外线和化学诱变剂引起的皮肤癌或与皮肤相关的疾病，从而改善人类的健康和福祉。

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. PUBLIC HEALTH RELEVANCE: DNA damage contributes to the development of 80-90% of human cancers. Ubiquitin- dependent proteolysis of DNA repair proteins is crucial for modulating functions of DNA damage recognition and removal in both normal cells and cancer cells. This study will characterize the roles of cullin 4A ubiquitin ligase in controlling the stability of damage sensors of the nucleotide excision repair apparatus. 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.