目的：基因组不稳定是癌症、免疫系统疾病、神经退行性病变等多种疾病的诱发因素。细胞为维持基因组稳定性，在长期进化过程中形成复杂的DNA损伤修复机制。细胞的DNA损伤修复机制是肿瘤放射治疗和化疗的理论基础。ATR是DNA双链断裂（DNA double strand break，DSB）修复过程中的重要激酶。它启动DNA损伤应答、促进DSB修复进程、调控细胞周期阻滞等。然而，ATR如何感应DSB，ATR活化如何受到其它蛋白的调控等很多重要问题仍未阐明。

NRF2蛋白通过调控细胞内氧化还原平衡和药物泵出等机制保护细胞。电离辐射条件下，NRF2可以通过调节细胞内活性氧（reactive oxygen species，ROS）水平维持基因组稳定性。近来有研究显示，NRF2在DNA损伤修复过程中也发挥重要作用，但具体机制仍不明确。

本研究将探索NRF2在DNA损伤修复过程中的新机制，建立NRF2与ATR之间的联系，为探究疾病的发病机制提供理论依据，为提高肿瘤放、化疗效果提供新途径。

方法： 

1：应用CRISPR/Cas9技术构建NRF2敲除细胞系和回补外源NRF2的细胞系。用siRNA或慢病毒敲降细胞内NRF2构建细胞模型。用DCFH-DA探针结合流式细胞术验证NAC清除ROS效果后，通过克隆形成实验、凋亡检测验证NRF2非依赖于ROS调控细胞对IR的敏感性。γH2AX和53BP1焦点免疫荧光实验结合CPT作用于细胞的克隆形成实验验证NRF2非依赖于ROS参与DSB修复。通过IR后细胞微核实验检测NRF2对基因组稳定性的影响。

2：通过Western Blotting实验检测细胞IR后NRF2对BRCA1、RAD51、53BP1等总蛋白和磷酸化水平的影响。通过免疫荧光实验检测IR或CPT处理细胞后BRCA1、RAD51焦点形成情况以及BRCA1焦点与53BP1焦点竞争情况。使用DR-GFP-U2OS HR报告系统检测NRF2对HR修复效率的影响。综合以上实验结果判断NRF2对HR修复方式是否有促进作用。

3：通过PI染色后流式细胞术检测IR或CPT处理细胞后NRF2对细胞周期分布的影响。另外，通过CyclinA免疫荧光实验和Western Blotting实验辅助证明NRF2对细胞周期阻滞的影响。

4：通过Western Blotting实验检测IR或CPT处理细胞后NRF2、ATR、ATM、p-ATR（Ser428）、p-ATM（Ser1981）、CHK1、p-CHK1（Ser317）、p-CHK1（Ser345）、p-CDC2（Tyr15）、CDC2、CyclinA等蛋白水平的变化判断NRF2通过ATR-CHK1-CDC2- CyclinA信号通路调控G2期细胞周期阻滞。

5：通过NRF2与TOPBP1、NRF2与ETAA1氨基酸序列比对推测NRF2对ATR活化的调控作用。通过外源表达野生和突变型NRF2蛋白检测NRF2促进ATR活化对AAD样结构域的依赖性。通过NRF2与γH2AX、NRF2与ATR共定位免疫荧光实验验证NRF2与ATR在DSB损伤位点同时形成焦点。通过ATR-NRF2蛋白质免疫共沉淀检测NRF2与ATR可以形成复合物。通过GST pull down实验检测NRF2与ATR体外结合情况，并检测NRF2与ATR结合对AAD样结构域的依赖性。

6：移植瘤模型中检测NRF2抑制剂Brusatol对IR抑瘤率的影响。通过Western Blotting实验检测NRF2、p-ATR（Ser428）、p-CHK1（Ser317）蛋白水平。通过肿瘤组织免疫荧光实验检测NRF2蛋白水平变化和p-ATR（Ser428）焦点形成。通过TUNEL染色检测IR后细胞凋亡。综合以上实验结果说明移植瘤模型中NRF2抑制剂通过ATR-CHK1信号通路增强放疗效果。

结果：

1：NRF2通过非依赖于调控ROS的新途径影响辐射敏感性和基因组稳定性；

2：NRF2在DSB修复过程中促进HR；

3：NRF2调控DSB修复进程中的G2期细胞周期阻滞；

4：NRF2通过活化ATR-CHK1信号通路促进DSB诱导的G2期细胞周期阻滞；

5：NRF2通过与ATR形成复合物调控ATR磷酸化；

6：移植瘤模型中NRF2抑制剂Brusatol具有放疗增敏作用。

结论：在IR或CPT诱导DSB条件下，NRF2在DSB损伤位点与ATR形成复合物；促进ATR-CHK1信号通路活化；促进G2期细胞周期阻滞；提高HR修复效率；维持基因组稳定性。

Objective： Genomic instability is the cause of cancer, immune system diseases, neurodegenerative diseases and other diseases. In order to maintain genomic stability, cells form complex DNA damage repair mechanisms in the long evolutionary process. The repair mechanism of DNA damage in cells is the theoretical basis of radiotherapy and chemotherapy. ATR is an important kinase in DNA double strand break (DSB) repair. It initiates DNA damage response, promotes DSB repair, and regulates cell cycle arrest. However, how ATR senses DSB, how ATR activation is regulated by other proteins and many other important issues have not been clarified. NRF2 protein protects cells by regulating intracellular redox balance and drug pumping. Under the condition of ionizing radiation, NRF2 can maintain genomic stability by regulating the level of intracellular reactive oxygen species (ROS). Recent studies have shown that NRF2 also plays an important role in DNA damage repair, but the specific mechanism is still unclear.

This project will explore the new mechanism of NRF2 in the process of DNA damage repair, establish the relationship between NRF2 and ATR, provide theoretical basis for exploring the pathogenesis of diseases, and provide a new way to improve the effect of radiotherapy and chemotherapy on tumors.

Methods:

1：The cell lines of NRF2 knockout and exogenous NRF2 were constructed by using the CRISPR/Cas9 technique. Cell models were constructed by knocking down intracellular NRF2 with siRNA or lentivirus. After the ROS scavenging effect of NAC was verified by DCFH-DA probe combined with flow cytometry, the sensitivity of cells not dependent on ROS to IR was verified by cloning formation assay and apoptosis detection. γH2AX and 53BP1 foci immunofluorescence assay combined with cloning formation assay of cells treated by CPT confirmed that NRF2 participated in DSB repair independent on regulation of ROS. The effect of NRF2 on genomic stability was detected by cell micronucleus assay after cells were exposed to IR.

2：The effects of NRF2 on the total protein and phosphorylation levels of BRCA1, RAD51, 53BP1, etc. were detected by Western Blotting, after cells were exposed to IR. The formation of BRCA1 and RAD51 foci and the competition between BRCA1 foci and 53BP1 foci were detected by immunofluorescence assay after IR or CPT treatment. The influence of NRF2 on HR repair efficiency was detected by using DR-GFP-U2OS HR reporting system. Based on the above results, we can judge whether NRF2 promotes HR repair.

3：The effect of NRF2 on cell cycle distribution after IR or CPT treatment was detected by flow cytometry after PI staining. In addition, the effect of NRF2 on cell cycle arrest was verified by CyclinA immunofluorescence assay and Western Blotting assay.

4：Western Blotting assay was used to detect the changes of NRF2, ATR, ATM, p-ATR(Ser428), p-ATM (Ser1981), CHK1, p-CHK1 (Ser317), p-CHK1 (Ser345), p-CDC2 (Tyr15), CDC2, CyclinA et al. protein levels after IR or CPT treatment. Then NRF2 was determined to regulate G2 cell cycle arrest through ATR-CHK1-CDC2- CyclinA signaling pathway.

5：The regulation of NRF2 on ATR activation was predicted by the amino acid sequence alignment between NRF2 and TOPBP1, NRF2 and ETAA1. The dependence of NRF2 on ATR activation on the AAD-like domain was detected by exogenous expression of wild type and mutant NRF2 proteins. The co-localization of NRF2 with γH2AX and NRF2 with ATR by immunofluorescence experiments verified that NRF2 and ATR simultaneously formed the foci at the DSB damage site. The formation of NRF2 complex with ATR was detected by immunoprecipitation of ATR-NRF2. In vitro binding of NRF2 to ATR was detected by GST pull down assay, and the dependence of NRF2 to ATR on AAD-like domain was detected.

6：The effect of NRF2 inhibitor Brusatol on IR tumor inhibition was detected in the transplanted tumor model. The protein levels of NRF2, p-ATR (Ser428) and p-CHK1 (Ser317) were detected by Western Blotting. Changes in NRF2 protein levels and p-ATR (Ser428) foci formation were detected by immunofluorescence assay in tumor tissues. Apoptosis after IR was detected by TUNEL staining. Combined with the above results, NRF2 inhibitors in the tumor model of transplantation enhance the radiotherapy effect through ATR-CHK1 signaling pathway.

Results:

1：NRF2 affects radiation sensitivity and genomic stability through a novel mechanism that is not dependent on ROS regulation;

2：NRF2 promotes HR in the repair process of DSB;

3：NRF2 regulates G2 cell cycle arrest in DSB repair process;

4：NRF2 promotes DSB-induced G2 cell cycle arrest by activating ATR-CHK1 signaling pathway.

5：NRF2 regulates ATR phosphorylation by forming a complex with ATR;

6：In the transplanted tumor model, the NRF2 inhibitor Brusatol has the effect of radiotherapy sensitization.

Conclusions：Under the condition of IR or CPT induced DSB, NRF2 formes a complex with ATR at the damage site of DSB, promotes the activation of ATR-CHK1 signaling pathway, promotes G2 cell cycle arrest, improves HR repair efficiency, maintains genomic stability.