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

PARP-1在哺乳动物转录偶联NER（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，其去除由不同试剂引起的各种损伤。NER有两个子途径：转录偶联NER（TCR）从活性基因的转录链上快速去除病变，而全局基因组NER（GGR）从基因组的其余部分缓慢去除病变。在这个NSERC支持的项目中，我们以前已经确定了哺乳动物核酶聚（ADP-核糖）聚合酶-1（PARP 1）在GGR中的两个关键作用。在这里，我们将重点讨论它在TCR中的作用。初步结果和假设：当转录RNA聚合酶II（RNAPII）在DNA损伤处停滞并招募Cockayne综合征B（CS B）蛋白开始多步过程以修复损伤并重新启动转录时，TCR启动。使用各种细胞TCR模型，我们现在已经观察到：（i）受损的PARP 1使细胞对由TCR修复的DNA损伤敏感;（ii）PARP 1在其遇到DNA损伤之前和之后都与RNAPII相互作用，但其仅在DNA损伤处与CSB相互作用;和（iii）在PARP 1受损的细胞中，CSB与RNAPII的相互作用（启动TCR所需的步骤）被显著抑制。 因此，我们的第一个假设是PARP 1在正常转录延伸期间与RNAPII一起存在，当RNAPII在损伤部位停滞时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PARP 1在TCR之前和之后的转录调节中发挥作用，以分别促进病变检测和转录重启。 为了检验该假设，直接目标是：1）阐明PARP 1与延伸的RNAPII和CSB在TCR起始中的作用; 2）检查PARP 1在DNA损伤后的从头转录中的作用，其在病变检测和TCR后转录的重新启动中起作用。方法和途径：我们建立了几种细胞和DNA损伤模型来研究TCR或GGR。将使用PARP 1基因敲除或敲低、酶失活PARP 1和PARP 1抑制剂检查PARP 1功能的影响。将通过Click-IT技术监测转录和TCR，以进行TCR特异性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PARP 1在TCR和所有基因转录中的全局影响。 长期目标是确定PARP 1在TCR中的干预是否在其他DNA修复途径中普遍观察到，这些DNA修复途径消除了基因转录链上不同类型的转录阻断病变。 影响：我们的研究计划旨在鉴定PARP 1在转录和TCR中的新作用，这不仅将提高我们对这些重要过程的教科书水平的知识，而且还可以更好地理解与TCR失败相关的疾病。

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.