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，可消除多种DNA损伤，包括紫外线（UV）诱导的DNA损伤。尽管对低等生物的NER通路进行了广泛的研究，但我们仍远未了解有效执行哺乳动物NER的全部蛋白质和过程。我们的研究计划将确定和表征聚（adp -核糖）聚合酶-1 （PARP-1）在哺乳动物NER中的不同作用，PARP-1是细菌或酵母中不存在的高等生物的核酶。***我们早期的研究表明，在细胞的紫外线照射下，PARP-1被催化激活形成adp核糖聚合物，这一过程在修复来自病毒报告基因或哺乳动物细胞基因组的紫外线诱导的DNA损伤中起作用。我们和其他人最近的研究发现，PARP-1通过与紫外线dna损伤结合蛋白2 （UV-DNA damage binding protein 2, DDB2）的相互作用和修饰，首次直接干预哺乳动物NER。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在体内紫外线诱导的DNA损伤部位的招募，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，这将使其能够与其他NER蛋白在损伤部位合作。****基于我们最近的数据，我们假设PARP-1在NER中有多种额外的作用，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)，它们分别在控制NER的全球基因组和转录偶联亚通路（GG-NER和TC-NER）的初始事件中起关键作用。我们还假设，紫外线损伤DNA激活PARP-1和PARylation影响各种NER蛋白的泛素化，从而促进NER蛋白的染色质重塑和运动，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的相互作用和控制功能，确定和表征PARP-1在GG-NER中的新作用，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是受损部位停滞的RNA聚合酶II启动TC-NER过程最早募集的蛋白；***3)研究PARP-1和PARylation是否控制不同E3泛素连接酶复合物参与NER蛋白泛素化，促进GG-NER和TC-NER的功能。******NER通路是一个很好的复杂模型，用于研究这种多用途且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。

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.