Regulation of Nucleotide Excision Repair by Proteolysis

摘要 核苷酸切除修复（NER）是主要的DNA修复机制，可以消除由DNA损伤引起的DNA损伤。 紫外线（UV）和化学诱变剂以防止基因组不稳定性和肿瘤发生。而 用于切除和修复DNA光损伤的酶促反应被充分研究，调节途径 对DNA损伤识别的时间和空间控制仍然知之甚少， 这种调节对肿瘤抑制的生理功能尚未被探索， 动物模型的不可用性。cullin 4A（CUL-4A）泛素连接酶最近成为一个关键的 两种DNA损伤传感器的调节因子：受损的DNA结合蛋白（DDBs，DDB 1和DDB 2的异二聚体）， DDB 2）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。期间 在上一个资助期，我们的生物化学和结构生物学研究提供了对 CUL-4A-DDB复合物的组装，以及c-Abl在活化细胞中的新的激酶非依赖性功能。 在正常条件下和UV照射后，DDBs的CUL-4A依赖性泛素化。重要的是， 我们产生了条件性CUL-4A基因敲除小鼠，并显示皮肤特异性CUL-4A基因敲除小鼠， 抗紫外线诱导的皮肤致癌作用，这表明一个有趣的药理学抑制的可能性 CUL-4A作为紫外线诱导的皮肤癌的预防策略。我们还与Stephan Goff博士合作， 确定DDB 1在NER中的生理功能，以及在控制细胞周期和基因组完整性中的生理功能。 条件DDB 1敲除小鼠。有趣的是，我们的体内研究揭示了细胞周期蛋白依赖性的 激酶抑制剂p21/CIP 1/WAF 1在CUL-4A-/-和DDB 1-/-小鼠以及MEF细胞中的作用， 角质形成细胞来源于这些小鼠。我们的长期目标是了解泛素途径 调节DNA修复并影响肿瘤发展。我们假设CUL-4A泛素连接酶 精确地控制活性，以确保NER的正确执行，并停止细胞周期事件， 有效修复的时间。我们处于独特的地位来测试这一假设，因为我们已经产生了具体的 泛素化抗性DDB 2突变体，鉴定了CUL-4A时间控制中的新调节剂（BRAP 2） 活性，并且具有CUL-4A、DDB 1和p21（或CIP 1或WAF 1）敲除小鼠。 我们建议采用生物化学、遗传学和细胞生物学方法相结合的方法来解决 具体目标有三：（1）建立CUL-4A泛素化调控DDB 2的机制 损伤识别和修复;（2）通过以下方法确定CUL-4A泛素连接酶活性的时间控制： （3）探讨p21在NER过程中积聚的机制基础和功能意义。 保护CUL-4A缺陷小鼠免受UV诱导的致癌作用。成功实现这些目标将 对我们理解泛素-蛋白酶体的分子和遗传基础有重要贡献 在DNA修复和肿瘤发生中起重要作用。从这些努力中获得的知识可以用来设计 用于预防和/或治疗UV和化学诱变剂诱导的皮肤癌或与皮肤相关的皮肤癌的新策略 疾病，从而改善人类的健康和福祉。

ABSTRACT 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. 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 cyclindependent 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 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 CUL-4A ubiquitination of DDB2 regulates damage recognition and repair; (2) To determine the temporal control of CUL-4A 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 skinrelated disorders, and thus improve the health and well-being of humans.