Multiple roles of poly(ADP-ribose) polymerase-1 in mammalian nucleotide excision repair

聚（ADP-核糖）聚合酶-1在哺乳动物核苷酸切除修复中的多重作用

• 批准号: RGPIN-2016-05868
• 负责人: Shah, Girish
• 金额: $ 2.77万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=664804
• 关键词: Multiple; roles; poly; ADP; ribose

Multiple Roles of PARP-1 in Mammalian NER***Background: Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions including those induced by ultraviolet radiations (UV). Despite extensive studies with lower organisms on NER pathways, we are still far from understanding the full complement of proteins and processes involved in efficiently executing mammalian NER. Our research program will identify and characterize different roles of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme of higher organisms that is absent in bacteria or yeast, in mammalian NER. ***Our earlier studies have shown that PARP-1 is catalytically activated to form polymers of ADP-ribose in response to UV-irradiation of cells and this process plays a role in the repair of UV-induced DNA damage from viral reporter genes or from the genome of mammalian cells. More recent studies from others and our team identified the first direct mechanism of intervention of PARP-1 in mammalian NER via its interaction with and modification of UV-DNA damage binding protein 2 (DDB2), a key lesion recognition protein of NER. Recently, we also developed two novel techniques to study recruitment of PARP-1 to UV-induced DNA lesion site in vivo and identified the bilateral asymmetric footprint of PARP-1 around UV-induced DNA lesion that would permit it to collaborate with other NER proteins at the lesion site.****Based on our recent data, we hypothesize that PARP-1 has multiple additional roles in NER, specifically through its interaction with two NER proteins, namely Xeroderma pigmentosum C (XPC) and Cockayne syndrome B (CSB), each of which play a key role in controlling the initial events in the global genomic and transcription-coupled sub-pathways of NER (GG-NER and TC-NER), respectively. We also hypothesize that PARP-1 activation by UV-damaged DNA and PARylation affects ubiquitination of various NER proteins, which facilitates chromatin remodelling and movement of NER proteins to efficiently conduct the repair events. ****To test our hypotheses, three immediate goals of our program are:***1) To identify and characterize novel role of PARP-1 in the GG-NER via its interaction with and control of the function of XPC, which starts the process of repair by GG-NER;***2) To determine whether PARP-1 influences the function of CSB, which is the earliest protein recruited by the stalled RNA polymerase II at the damaged site for initiating the process of TC-NER;***3) To examine whether PARP-1 and PARylation control the functions of different E3 ubiquitin ligase complexes involved in ubiquitination of NER proteins, which facilitate GG-NER and TC-NER.******NER pathway serves as an excellent complex model to examine multiple roles of this versatile and abundant yet enigmatic protein in mammalian cells, and our long-term goal is to apply this knowledge to understand various other functions of PARP-1 in mammalian cells.

PARP-1在哺乳动物NER中的多重作用 * 背景：核苷酸切除修复（NER）是最通用的DNA修复途径，其去除各种DNA损伤，包括由紫外线辐射（UV）诱导的DNA损伤。尽管广泛的研究与较低的生物体的NER途径，我们仍然远远没有了解的蛋白质和过程中涉及的有效执行哺乳动物NER的完整补充。我们的研究计划将确定和表征聚（ADP-核糖）聚合酶-1（PARP-1），一种在细菌或酵母中不存在的高等生物的核酶，在哺乳动物NER中的不同作用。* 我们早期的研究已经表明，PARP-1被催化活化以响应于细胞的UV照射形成ADP-核糖的聚合物，并且该过程在修复来自病毒报告基因或来自哺乳动物细胞基因组的UV诱导的DNA损伤中起作用。其他人和我们团队最近的研究确定了PARP-1干预哺乳动物NER的第一个直接机制，通过其与UV-DNA损伤结合蛋白2（DDB 2）的相互作用和修饰，DDB 2是NER的关键损伤识别蛋白。 最近，我们还开发了两种新技术来研究PARP-1在体内UV诱导的DNA损伤部位的募集，并确定了PARP-1在UV诱导的DNA损伤周围的双侧不对称足迹，这将允许它与损伤部位的其他NER蛋白合作。基于我们最近的数据，我们假设PARP-1在NER中具有多种额外的作用，特别是通过其与两种NER蛋白的相互作用，即着色性干皮病C（XPC）和Cockayne综合征B（CS B），每种蛋白分别在控制NER的全局基因组和转录偶联子途径（GG-NER和TC-NER）中的初始事件中发挥关键作用。我们还假设，由UV损伤的DNA和PAR化激活的PARP-1影响各种NER蛋白的泛素化，这有助于染色质重塑和NER蛋白的运动，以有效地进行修复事件。* 为了验证我们的假设，我们计划的三个直接目标是：*1）鉴定和表征PARP-1在GG-NER中的新作用，其通过与XPC的相互作用和控制XPC的功能，启动GG-NER的修复过程;*2）确定PARP-1是否影响CSB的功能，CSB是被停滞的RNA聚合酶II在损伤位点招募的最早蛋白，用于启动TC-NER过程;*3）检查PARP-1和PAR化是否控制参与NER蛋白泛素化的不同E3泛素连接酶复合物的功能，其促进GG-NER和TC-NER。NER通路是一个很好的复杂模型，可以研究这种多功能，丰富但神秘的蛋白质在哺乳动物细胞中的多种作用，我们的长期目标是应用这些知识来了解哺乳动物细胞中PARP-1的各种其他功能。