Roles of poly(ADP-ribose) polymerase-1 in mammalian transcription-coupled nucleotide excision repair of DNA damage

聚（ADP-核糖）聚合酶-1在哺乳动物转录偶联核苷酸切除修复DNA损伤中的作用

• 批准号: RGPIN-2022-05355
• 负责人: Shah, Girish
• 金额: $ 2.33万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745061
• 关键词: Roles; poly; ADP; ribose; polymerase

Roles of PARP-1 in mammalian transcription-coupled-NER (TCR) of DNA damage Background: Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of lesions caused by diverse agents. There are two sub-pathways of NER: the transcription-coupled NER (TCR) rapidly removes lesions from the transcribed strand of the active genes, whereas the global genomic NER (GGR) slowly removes the lesions from rest of the genome. In this NSERC-supported program, we have previously identified two key roles of the mammalian nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) in GGR. Here, we will focus on its role in TCR. Preliminary Results and Hypotheses: TCR is initiated when the transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits the Cockayne syndrome B (CSB) protein to begin a multistep process to repair the damage and restart the transcription. Using various cellular TCR models, we have now observed that: (i) Impaired PARP1 sensitizes cells to DNA damage that is repaired by TCR; (ii) PARP1 interacts with RNAPII both before and after it encounters DNA damage, but it interacts with CSB only at the DNA damage; and (iii) In PARP1-impaired cells, the interaction of CSB with RNAPII, a required step to launch TCR, is significantly suppressed. Therefore, our first hypothesis is that PARP1 present with RNAPII during normal transcription elongation, switches to a repair role when RNAPII stalls at the lesion site to facilitate efficient initiation of TCR. Our second hypothesis is that PARP1 plays a role in transcription regulation before and after TCR to facilitate lesion detection and transcription restart, respectively. To test the hypothesis, the immediate goals are to: 1) Elucidate the role of PARP1 with the elongating RNAPII and CSB in the initiation of TCR; 2) Examine the role of PARP1 in the de novo transcription after DNA damage that plays a role in the lesion detection and in the restart of transcription after TCR. Methods and Approaches: We have established several cellular and DNA damage models to study the TCR or GGR. The impact of PARP1 function will be examined using PARP1-gene knockout or knockdown, enzyme-dead PARP1 and PARP-inhibitors. The transcription and TCR will be monitored by Click-IT techniques for TCR-specific RNA recovery synthesis and unscheduled DNA synthesis. We will use ChIP-Seq and BrU-Seq techniques to identify global impact of PARP1 in TCR and transcription of all genes. The long-term objective is to determine if the intervention of PARP1 in TCR is universally observed in other DNA repair pathways that remove different types of transcription-blocking lesions on the transcribed strand of genes. Impact: Our research program aimed at identification of novel roles of PARP1 in transcription and TCR will not only advance our text-book level knowledge of these important processes but also allow better comprehension of the diseases associated with failure of TCR.

PARP-1在哺乳动物转录偶联修复(TCR)DNA损伤中的作用背景：核苷酸切除修复(NER)是最通用的DNA修复途径，可以去除由不同因素引起的各种损伤。NER有两个子途径：转录偶联NER(TCR)快速去除活性基因转录链上的病变，而全局基因组NER(GGR)缓慢去除基因组其余部分的病变。在这个由NSERC支持的计划中，我们先前已经确定了哺乳动物核酶多聚(ADP-核糖)聚合酶-1(PARP1)在GGR中的两个关键作用。在这里，我们将重点介绍它在TCR中的作用。初步结果和假设：TCR是当转录RNA聚合酶II(RNAPII)在DNA损伤处停止并招募Cockayne综合征B(CSB)蛋白开始一个多步骤的过程来修复损伤并重新启动转录时启动的。使用不同的细胞TCR模型，我们现在已经观察到：(I)PARP1受损的细胞对TCR修复的DNA损伤敏感；(Ii)PARP1在遇到DNA损伤之前和之后都与RNAPII相互作用，但它只在DNA损伤时与CSB相互作用；以及(Iii)在PARP1受损的细胞中，CSB与RNAPII的相互作用受到显著抑制，RNAPII是启动TCR的必要步骤。因此，我们的第一个假设是，在正常的转录延伸过程中，PARP1与RNAPII一起存在，当RNAPII在病变部位停滞时，PARP1切换到修复作用，以促进TCR的有效启动。我们的第二个假设是，PARP1在TCR前和TCR后分别参与转录调控，以促进病变检测和转录重新启动。为了验证这一假说，近期的目标是：1)阐明PARP1与延长的RNAPII和CSB在启动TCR中的作用；2)检测PARP1在DNA损伤后的从头转录中的作用，这在损伤检测和TCR后转录的重新启动中发挥作用。方法和方法：我们建立了多种细胞和DNA损伤模型来研究TCR或GGR。将使用PARP1基因敲除或敲除、酶死亡的PARP1和PARP抑制剂来检查PARP1功能的影响。转录和TCR将通过Click-IT技术进行监测，以进行TCR特异的RNA回收合成和非计划DNA合成。我们将使用ChIP-Seq和Bru-Seq技术来确定PARP1在TCR和所有基因转录中的全球影响。长期目标是确定PARP1在TCR中的干预是否普遍存在于其他DNA修复途径中，这些途径可以消除转录基因链上不同类型的转录阻断损伤。影响：我们旨在确定PARP1在转录和TCR中的新角色的研究计划不仅将促进我们对这些重要过程的教科书水平的知识，而且还将使我们能够更好地理解与TCR失败相关的疾病。