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

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，可以去除包括紫外线辐射(UV)在内的多种DNA损伤。尽管在NER途径上对低等生物进行了广泛的研究，但我们仍然远未了解有效执行哺乳动物NER所涉及的蛋白质和过程的全部补充。我们的研究计划将识别和表征聚(ADP-核糖)聚合酶-1(PARP-1)在哺乳动物NER中的不同作用。PARP-1是高等生物的一种核酶，在细菌或酵母中不存在。我们早期的研究表明，PARP-1在细胞的紫外线照射下被催化激活，形成ADP-核糖聚合物，这一过程在修复紫外线诱导的报告基因或哺乳动物细胞基因组的DNA损伤中发挥了作用。其他人和我们团队最近的研究发现了PARP-1通过与NER的关键损伤识别蛋白UV-DNA损伤结合蛋白2(DDB2)相互作用和修饰来干预哺乳动物NER的第一个直接机制。最近，我们还发展了两种新的技术来研究PARP-1在体内紫外线诱导的DNA损伤部位的募集，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蛋白协作。*根据我们最近的数据，我们假设PARP-1在NER中具有多个额外的作用，特别是通过它与两种NER蛋白的相互作用，即着色性干皮病C(XPC)和Cockayne综合征B(CSB)。它们中的每一个分别在NER(GG-NER和TC-NER)的全局基因组和转录偶联亚途径中起着关键的控制作用。我们还假设，紫外线损伤的DNA和PAR化激活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蛋白的移动，从而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)通过与XPC功能的相互作用和对XPC功能的控制，鉴定和表征PARP-1在GG-NER中的新角色，XPC启动了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在损伤部位招募的最早启动TC-NER过程的蛋白质；*3)检测PARP-1和PAR化是否控制参与NER蛋白泛素化的不同E3泛素连接酶复合体的功能，从而促进GG-NER和TC-NER。*NER途径是一个很好的复杂模型，可以研究这种功能广泛且丰富的神秘蛋白在哺乳动物细胞中的多种作用，我们的长期目标是应用这一知识来了解PARP-1在哺乳动物细胞中的其他各种功能。

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.