南方科技大学知识苑(SUSTech KC): DNA 聚合酶θ抑制剂的筛选和检测体系

题名	DNA 聚合酶θ抑制剂的筛选和检测体系
其他题名	SCREENING AND DETECTION SYSTEMS FOR DNA POLYMERASE THETA INHIBITORS
姓名	王佳
姓名拼音	WANG Jia
学号	12132796
学位类型	硕士
学位专业	0856 材料与化工
学科门类/专业学位类别	0856 材料与化工
导师	贾铁争
导师单位	化学系
外机构导师	邓麟
外机构导师单位	深圳湾实验室
论文答辩日期	2023-05-24
论文提交日期	2023-06-27
学位授予单位	南方科技大学
学位授予地点	深圳
摘要	为了维持基因组的稳定性，哺乳动物细胞已经进化出多种通路来修复双链 DNA 断裂，包括由 DNA 聚合酶θ（DNA polymerase θ，Polθ）主导的微同源介导的末端连接（Microhomology-mediated end-joining，MMEJ）通路。Polθ由 POLQ 基因编码，在多种肿瘤细胞中过表达，对同源重组缺陷型癌细胞的增殖至关重要。同时，PARP（Poly ADP-ribose Polymerase）抑制剂和 Polθ抑制剂具有协同作用，抑制 Polθ能有效地降低 PARP 抑制剂处理带来的耐药性。基于此，Polθ是近年来药物筛选的热门靶点。本文利用大肠杆菌蛋白表达体系，成功地纯化出 Polθ解旋酶结构域（Polθ-hel）和聚合酶结构域（Polθ-pol）蛋白，ADP-Glo 试验和 Picogreen 试验结果表明纯化的蛋白分别具有较高的解旋酶和聚合酶活性。通过这些高活性蛋白，本文可以在体外搭建高通量药物筛选体系，从而针对这两个结构域进行大规模药物筛选。同时，通过 CRISPR/Cas9 技术在 HEK293-FT细胞中，本文成功地构建了 GFP-Polθ诱导表达系统。荧光成像和 western blot 试验结果表明本文可以容易地获得大量表达 GFP-Polθ蛋白的细胞，这将为纯化 Polθ蛋白的全长打下基础；且利用人源细胞进行 Polθ蛋白纯化将表现出其他蛋白纯化系统所不具有的最大程度还原 Polθ蛋白结构和活性的优势。更进一步，本文利用 CRISPR/Cas9 技术在人 RPE-1 细胞中成功构建了 POLQ 和 BRCA2 基因的敲除细胞系。分别通过基因测序、PCR、qRT- PCR、免疫荧光、细胞活力和细胞增殖检测等方法进行了验证。BRCA2 基因敲除细胞系可通过合成致死效应来测试 Polθ抑制剂的细胞毒性；POLQ 基因敲除细胞系可检测 Polθ抑制剂的作用靶点是否具有专一性。本文中，Polθ抑制剂的高通量药物筛选体系的搭建和药物检测细胞模型的构建将为同源重组缺陷型疾病患者的治疗做出一定贡献。
其他摘要	Mammalian cells have developed various mechanisms to repair DNA double-strand breaks and maintain genomic stability. One such pathway is microhomology-mediated end-joining (MMEJ), which is led by DNA polymerase theta (DNA polymerase θ, Polθ). The POLQ gene encode Polθ, which is highly expressed in multiple cancer types and is essential for the proliferation of homologous recombination-deficient cancer cells. In addition, Polθ inhibitors have shown synergistic effects with PARP (Poly ADP-ribose Polymerase) inhibitors, as inhibiting Polθ can effectively reduce drug resistance induced by PARP inhibitor. Consequently, Polθ has emerged as a promising drug target in recent years. In this study, we were able to purify the structural domains of Polθ helicase and polymerase proteins using the E. coli protein expression system. The results of both the ADP-Glo and Picogreen assay indicated that the purified proteins exhibited significant helicase and polymerase activities, respectively. These highly active proteins can be utilized to construct an in vitro high-throughput drug screening systems, enabling large-scale drug screening against the two domains. Meanwhile, we successfully constructed an inducible expression system for GFP-Polθ using CRISPR/Cas9 technology in HEK293-FT cells. The results of fluorescence imaging and western blot assay demonstrated that large cells expressing GFP-Polθ protein can be easily obtained, which will facilitate the purification of the full-length Polθ protein. Using human-derived cells for Polθ protein purification offers an advantage over other protein purification systems. The GFP-Polθ protein purification system will maximally maintain the protein structure and activity of Polθ. Additionally, we successfully created knocked-out cell lines of POLQ and BRCA2 genes in human RPE-1 cells using CRISPR/Cas9 technology. The validation of the knocked-out cell lines was done using various methods such as gene sequencing, PCR, qRT-PCR, Immunofluorescence, cell viability and cell proliferation. The BRCA2 knocked-out cell lines were used to test the cytotoxicity of Polθ inhibitors through synthetic lethal effects, whereas the POLQ knocked-out cell lines were used to test the specificity. The development of a high-throughput drug screening system and drug detection cell model for Polθ inhibitors is a significant contribution towards treating patients with homologous recombination-deficient diseases.
关键词	DNA 聚合酶θ 抑制剂蛋白纯化合成致死
其他关键词	Polθ Inhibitors Protein Purification Synthetic Lethality
语种	中文
培养类别	独立培养
入学年份	2021
学位授予年份	2023-06
参考文献列表	[1] MASUD T, SOONG C, XU H, et al. Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461[J]. Scientific Reports, 2021, 11(1): 9812. [2] BELAN O, SEBALD M, ADAMOWICZ M, et al. POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells[J]. Molecular Cell, 2022, 82(24): 4664-4680 e4669. [3] PORUBSKY D, HOPS W, ASHRAF H, et al. Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders[J]. Cell, 2022, 185(11): 1986-2005 e1926. [4] STILLMAN B. Reconsidering DNA Polymerases at the Replication Fork in Eukaryotes[J]. Molecular Cell, 2015, 59(2): 139-141. [5] MUELLER S C, BACKES C, HAAS J, et al. Pathogenicity prediction of non-synonymous single nucleotide variants in dilated cardiomyopathy[J]. Briefings in Bioinformatics, 2015, 16(5): 769-779. [6] KENIRY M A, et al. NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli[J]. Protein Science, 2000, 9(4): 721-733. [7] JAIN R, AGGARWAL A K, RECHKOBLIT O. Eukaryotic DNA polymerases[J]. Current Opinion in Structural Biology, 2018, 53: 77-87. [8] RAO X, XING B, WU Z, et al. Targeting polymerase theta impairs tumorigenesis and enhances radiosensitivity in lung adenocarcinoma[J]. Cancer Science, 2023, 1: 1-4. [9] KENT T, CHANDRAMOULY G, MCDEVITT S M, et al. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase theta[J]. Nature Structural & Molecular Biology, 2015, 22(3): 230-237. [10] SFEIR A, SYMINGTON L S. Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway[J]. Trends in Biochemical Sciences, 2015, 40(11): 701-714. [11] LEMEE F, BERGOGLIO V, et al. DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(30): 13390-13395. [12] MATEOS，et al. The helicase domain of Poltheta counteracts RPA to promote alt-NHEJ[J]. Nature Structural & Molecular Biology, 2017, 24(12): 1116-1123. [13] FERNANDEZ-VIDAL A, et al. A role for DNA polymerase theta in the timing of DNA replication[J]. Nature Communication, 2014, 5: 4285. [14] KOSICKI, et al. Cas9-induced large deletions and small indels are controlled in a convergent fashion[J]. Nature Communication, 2022, 13(1): 3422. [15] CHEN X S, POMERANTZ R T. DNA Polymerase theta: A Cancer Drug Target with Reverse Transcriptase Activity[J]. Genes (Basel), 2021, 12(8). [16] CHANDRAMOULY G, ZHAO J, MCDEVITT S, et al. Poltheta reverse transcribes RNA and promotes RNA-templated DNA repair[J]. Science Advance, 2021, 7(24): 2256. [17] SEKI M, MARINI F, WOOD R D, et al. POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells[J]. Nucleic Acids Research, 2003, 31(21): 6117-6126. [18] WOOD R D, DOUBLIE S. DNA polymerase theta (POLQ), double-strand break repair, and cancer[J]. DNA Repair, 2016, 44: 22-32. [19] WANG Z, SONG Y, LI S, et al. DNA polymerase theta (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse[J]. Journal of Biological Chemistry, 2019, 294(11): 3909-3919. [20] YOUSEFZADEH M J, WOOD R D, et al. DNA polymerase POLQ and cellular defense against DNA damage[J]. DNA Repair, 2013, 12(1): 1-9. [21] DENG L, WU R A, SONNEVILLE R, et al. Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements[J]. Molecular Cell, 2019, 73(5): 915-929 e916. [22] YOON J H, et al. Error-Prone Replication through UV Lesions by DNA Polymerase theta Protects against Skin Cancers[J]. Cell, 2019, 176(6): 1295-1309 e1215. [23] BEAGAN K, MCVEY M, et al. Linking DNA polymerase theta structure and function in health and disease[J]. Cellular and Molecular Life Sciences, 2016, 73(3): 603-615. [24] SEKI M, WOOD R D, et al. DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct[J]. DNA Repair (Amst), 2008, 7(1): 119-127. [25] ZHENG D Q, PETES T D, et al. Genome Instability Induced by Low Levels of Replicative DNA Polymerases in Yeast[J]. Genes (Basel), 2018, 9(11). [26] KRAMARA J, OSIA B, MALKOVA A, et al. Break-Induced Replication: The Where, The Why, and The How[J]. Trends in Genetics, 2018, 34(7): 518-531. [27] GROELLY F J, FAWKES M, DAGG R A, et al. Targeting DNA damage response pathways in cancer[J]. Nature Reviews: Cancer, 2022, 5: 120-123. [28] BALDACCI G, et al. Impact of the DNA polymerase Theta on the DNA replication program[J]. Genome Data, 2015, 3: 90-93. [29] ARNOULT N, et al. Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN[J]. Nature, 2017, 549(7673): 548-552. [30] JACHIMOWICZ R D, GOERGENS J, REINHARDT H C, et al. DNA double-strand break repair pathway choice-from basic biology to clinical exploitation[J]. Cell Cycle, 2019, 18(13): 1423-1434. [31] SONG B, et al. Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage[J]. International Journal of Molecular Sciences, 2021, 22(12). [32] CHANG H H Y, PANNUNZIO N R, ADACHI N, et al. Non-homologous DNA end joining and alternative pathways to double-strand break repair[J]. Nature Reviews: Molecular Cell Biology, 2017, 18(8): 495-506. [33] WYATT D W, FENG W, CONLIN M P, et al. Essential Roles for Polymerase theta-Mediated End Joining in the Repair of Chromosome Breaks[J]. Molecular Cell, 2016, 63(4): 662-673. [34] ROSSI M J, DIDOMENICO S F, PATEL M, et al. RAD52: Paradigm of Synthetic Lethality and New Developments[J]. Front Genet, 2021, 12: 780293. [35] SCULLY R, PANDAY A, ELANGO R, et al. DNA double-strand break repair-pathway choice in somatic mammalian cells[J]. Nature Reviews: Molecular Cell Biology, 2019, 20(11): 698-714. [36] TRUONG L N, et al. Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(19): 7720-7725. [37] LLORENS-AGOST M, ENSMINGER M, LE H P, et al. POL theta-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis[J]. Nature Cell Biology, 2021, 23(10): 1095-1104. [38] CHANDRAMOULY G, JAMSEN J, BORISONNIK N, et al. Pollambda promotes microhomology-mediated end-joining[J]. Nature Structural & Molecular Biology, 2023, 30(1): 107-114. [39] LAVERTY D J, MORTIMER I P, GREENBERG M, et al. Mechanistic Insight through Irreversible Inhibition: DNA Polymerase theta Uses a Common Active Site for Polymerase and Lyase Activities[J]. Journal of the American Chemical Society, 2018, 140(29): 9034-9037. [40] CARVAJAL-GARCIA J, CROWN K N, et al. DNA polymerase theta suppresses mitotic crossing over[J]. PLos Genetics, 2021, 17(3): e1009267. [41] CAMPBELL J L, LI H, et al. Pol theta helicase: drive or reverse[J]. Nature Structural & Molecular Biology, 2017, 24(12): 1007-1008. [42] BLACK S J, KASHKINA E, KENT T, et al. DNA Polymerase theta: A Unique Multifunctional End-Joining Machine[J]. Genes (Basel), 2016, 7(9): 2214. [43] BRANDSMA I, et al. Pathway choice in DNA double strand break repair: observations of a balancing act[J]. Functional & Integrative Genomics, 2012, 3(1): 9. [44] KENNEDY R D, D'ANDREA A D, et al. DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes[J]. Journal of Clinical Oncology, 2006, 24(23): 3799-3808. [45] FARMER H, MCCABE N, et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy[J]. Nature, 2005, 434(7035): 917-921. [46] ZHAO, N., SONG, Y., XIE, X., ZHU, Z, et al. Synthetic biology-inspired cell engineering in diagnosis, treatment, and drug development[J]. Signal Transduct Target Therapy. 2023, 8, 112. [47] YU, W, et al. The Development of Drug Delivery Systems for Efficient Intracranial Hemorrhage Therapy[J]. Advance Healthy Mater. 2023, 12: e2203141. [48] MATEOS-GOMEZ P A, GONG F, NAIR N, et al. Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination[J]. Nature, 2015, 518(7538): 254-257. [49] GOULLET DE RUGY T, BASHKUROV M, DATTI A, et al. Excess Poltheta functions in response to replicative stress in homologous recombination-proficient cancer cells[J]. Biology Open, 2016, 5(10): 1485-1492. [50] WARD, R.M, et al. Improving Drug Therapy for Pediatric Patients: Unfinished History of Pediatric Drug Development[J]. J Pediatr Pharmacol Ther. 2023, 28: 4-9. [51] PUGH K W, ZHANG Z, et al. From Bacteria to Cancer: A Benzothiazole-Based DNA Gyrase B Inhibitor Redesigned for Hsp90 C-Terminal Inhibition[J]. ACS Medicinal Chemistry Letters, 2020, 11(8): 1535-1538. [52] HYUN S Y, LE H T, NGUYEN C T, et al. Development of a novel Hsp90 inhibitor NCT-50 as a potential anticancer agent for the treatment of non-small cell lung cancer[J]. Scientific Reports, 2018, 8(1): 13924. [53] ZHOU J, GELOT C, PANTELIDOU C, et al. A first-in-class Polymerase Theta Inhibitor selectively targets Homologous-Recombination-Deficient Tumors[J]. Nature Cancer, 2021, 2(6): 598-610. [54] ZATREANU D, ROBINSON H M R, ALKHATIB O, et al. Poltheta inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance[J]. Nature Communication, 2021, 12(1): 3636. [55] BUBENIK M, MADER P, MOCHIRIAN P, et al. Identification of RP-6685, an Orally Bioavailable Compound that Inhibits the DNA Polymerase Activity of Poltheta[J]. Journal of Medicinal Chemistry, 2022. [56] SHINMURA K, KATO H, KAWANISHI Y, et al. POLQ Overexpression Is Associated with an Increased Somatic Mutation Load and PLK4 Overexpression in Lung Adenocarcinoma[J]. Cancers, 2019, 11(5). [57] LI J, et al. Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma[J]. Cancers, 2021, 13(13):1256. [58] CECCALDI R, LIU J C, AMUNUGAMA R, et al. Homologous-recombination-deficient tumours are dependent on Poltheta-mediated repair[J]. Nature, 2015, 518(7538): 258-262. [59] SIMSEK D, JASIN M et al. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation[J]. Nature Structural & Molecular Biology, 2010, 17(4): 410-U443. [60] MAH LJ, EL-OSTA A, KARAGIANNIS TC, et al. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia[J]. 2010 Apr;24(4):679-86. [61] KUO LJ, YANG LX, et al. Gamma-H2AX - a novel biomarker for DNA double-strand breaks. In Vivo[J]. 2008 May-Jun;22(3):305-9. PMID: 18610740. [62] BYRNE BM, OAKLEY GG, et al. Replication protein A, the laxative that keeps DNA regular: The importance of RPA phosphorylation in maintaining genome stability[J]. Semin Cell Developmental Biology. 2019; 86:112-120. [63] IFTODE C, DANIELY Y, et al. Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochemistry Molecular Biology[J]. 1999;34(3):141-80. [64] SCHREMPF A, BERNARDO S, et al. POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells[J]. Cell Report. 2022 Nov 29;41(9):111716. [65] MOMOZAWA Y, SASAI R, USUI Y, et al. Expansion of Cancer Risk Profile for BRCA1 and BRCA2 Pathogenic Variants[J]. JAMA Oncology. 2022 Jun 1;8(6):871-878. [66] MANN A, RAMIREZ-OTERO MA, DE ANTONI A, et al. POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps[J]. Molecular Cell. 2022 Nov 17;82(22):4218-4231.e8. [67] DAVIES H, GLODZIK D, MORGANELLA S, et al. HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures[J]. Nature Medicine. 2017 Apr;23(4):517-525.
所在学位评定分委会	化学
国内图书分类号	Q512
来源库	人工提交
成果类型	学位论文
条目标识符	http://sustech.caswiz.com/handle/2SGJ60CL/544433
专题	理学院_化学系
推荐引用方式 GB/T 7714	王佳. DNA 聚合酶θ抑制剂的筛选和检测体系[D]. 深圳. 南方科技大学,2023.

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