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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710007
• 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，可消除多种 DNA 损伤，包括由紫外线 (UV) 引起的损伤。尽管对低等生物体的 NER 通路进行了广泛的研究，但我们仍然远未了解有效执行哺乳动物 NER 所涉及的全部蛋白质和过程。我们的研究计划将鉴定和表征多聚（ADP-核糖）聚合酶-1 (PARP-1) 在哺乳动物 NER 中的不同作用，PARP-1 是一种高等生物的核酶，细菌或酵母中不存在。 我们早期的研究表明，响应细胞的紫外线照射，PARP-1 被催化激活形成 ADP-核糖聚合物，这一过程在修复紫外线诱导的病毒报告基因或哺乳动物细胞基因组 DNA 损伤中发挥着重要作用。其他人和我们团队最近的研究确定了 PARP-1 通过与 UV-DNA 损伤结合蛋白 2 (DDB2) 相互作用并对其进行修饰来干预哺乳动物 NER 的第一个直接机制，DDB2 是 NER 的关键病变识别蛋白。 最近，我们还开发了两种新技术来研究 PARP-1 在体内招募到紫外线诱导的 DNA 损伤部位的情况，并确定了 PARP-1 在紫外线诱导的 DNA 损伤周围的双边不对称足迹，这将使其能够与损伤部位的其他 NER 蛋白合作。 根据我们最近的数据，我们假设 PARP-1 在 NER 中具有多种附加作用，特别是通过它与两种 NER 蛋白相互作用，即着色性干皮病 C (XPC) 和科凯恩综合征 B (CSB)，这两种蛋白分别在控制 NER 的全局基因组和转录偶联子通路（GG-NER 和 TC-NER）的初始事件中发挥着关键作用。我们还假设 UV 损伤的 DNA 和 PARylation 激活 PARP-1 会影响各种 NER 蛋白的泛素化，从而促进染色质重塑和 NER 蛋白的运动，从而有效地进行修复事件。 为了检验我们的假设，我们计划的三个直接目标是： 1) 通过与 XPC 的相互作用和控制 XPC 的功能来识别和表征 PARP-1 在 GG-NER 中的新作用，XPC 启动 GG-NER 的修复过程； 2) 确定PARP-1是否影响CSB的功能，CSB是停滞的RNA聚合酶II在受损位点招募的最早的蛋白质，用于启动TC-NER过程； 3) 检查PARP-1和PARylation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，从而促进GG-NER和TC-NER。 NER 通路作为一个优秀的复杂模型来检查这种多功能、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 PARP-1 在哺乳动物细胞中的各种其他功能。