Regulation of Nucleotide Excision Repair by Proteolysis

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，可去除紫外线（UV）和化学诱变剂引起的DNA损伤，以防止基因组不稳定和肿瘤发生。虽然DNA光损伤的切除和修复的酶促反应已经得到了很好的研究，但调控DNA损伤识别的时空控制的调控途径仍然知之甚少，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被探索。cullin 4A （CUL-4A）泛素连接酶最近被发现是两种DNA损伤传感器的关键调节剂：受损的DNA结合蛋白（DDBs， DDB1和DDB2的异源二聚体）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。在之前的资助期内，我们的生化和结构生物学研究提供了CUL-4A-DDB复合物组装的机制，以及c-Abl在正常条件和紫外线照射下激活cul - 4a依赖的ddb泛素化的新激酶独立功能。重要的是，我们培育了条件CUL-4A敲除小鼠，并发现皮肤特异性CUL-4A敲除小鼠对紫外线诱导的皮肤癌具有抗性，这表明药理抑制CUL-4A可能是一种预防紫外线诱导皮肤癌的策略。虽然CUL-4B在细胞生长和存活方面与CUL-4A具有重叠的功能，但其在DDB2降解和NER中的作用似乎不如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，我们的体内研究显示，在CUL-4A-/-和DDB1-/-小鼠以及MEF细胞和源自这些小鼠的角化细胞中，周期蛋白依赖性激酶抑制剂p21/CIP1/WAF1显著上调。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验证这一假设，因为我们已经产生了特异性的泛素化抗性DDB2突变体，发现了一种新的调节剂（BRAP2），可以在紫外线照射后暂时控制CUL-4A的活性，并且已经拥有了CUL-4A、DDB1和p21（或CIP1或WAF1）敲除小鼠。我们建议采用生物化学、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BRAP2在NER期间对CUL-4A和CUL-4B泛素连接酶活性的时间调控；(3)确定p21积累在保护cul - 4a缺陷小鼠免受紫外线致癌作用中的机制基础和功能意义。这些目标的成功完成将大大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基础。从这些努力中获得的知识可用于制定预防和/或治疗紫外线和化学诱变剂引起的皮肤癌或与皮肤有关的疾病的新战略，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.