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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Yi-Wen Liao | en |
dc.contributor.author | 廖怡雯 | zh_TW |
dc.date.accessioned | 2021-06-15T16:08:26Z | - |
dc.date.available | 2018-09-25 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-19 | |
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Nat Rev Cancer 9, 327-337, (2009). 9 Pommier, Y., Leo, E., Zhang, H. & Marchand, C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol 17, 421-433, (2010). 10 Champoux, J. J. DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70, 369-413, (2001). 11 Corbett, K. D. & Berger, J. M. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct 33, 95-118, (2004). 12 Vos, S. M., Tretter, E. M., Schmidt, B. H. & Berger, J. M. All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12, 827-841, (2011). 13 Gadelle, D., Filee, J., Buhler, C. & Forterre, P. Phylogenomics of type II DNA topoisomerases. Bioessays 25, 232-242, (2003). 14 Sissi, C. & Palumbo, M. Effects of magnesium and related divalent metal ions in topoisomerase structure and function. Nucleic Acids Res 37, 702-711, (2009). 15 Corbett, K. D., Schoeffler, A. J., Thomsen, N. D. & Berger, J. M. The structural basis for substrate specificity in DNA topoisomerase IV. J Mol Biol 351, 545-561, (2005). 16 Bergerat, A., Gadelle, D. & Forterre, P. Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae. A thermostable enzyme with both bacterial and eucaryal features. J Biol Chem 269, 27663-27669 (1994). 17 Hartung, F. et al. An archaebacterial topoisomerase homolog not present in other eukaryotes is indispensable for cell proliferation of plants. Curr Biol 12, 1787-1791 (2002). 18 Nichols, M. D., DeAngelis, K., Keck, J. L. & Berger, J. M. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11. EMBO J 18, 6177-6188, (1999). 19 Keeney, S. Mechanism and control of meiotic recombination initiation. Curr Top Dev Biol 52, 1-53 (2001). 20 Schoeffler, A. J. & Berger, J. M. Recent advances in understanding structure-function relationships in the type II topoisomerase mechanism. Biochem Soc Trans 33, 1465-1470, (2005). 21 Schmidt, B. H., Osheroff, N. & Berger, J. M. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity. Nat Struct Mol Biol 19, 1147-1154, (2012). 22 Chang, C. C., Wang, Y. R., Chen, S. F., Wu, C. C. & Chan, N. L. New insights into DNA-binding by type IIA topoisomerases. Curr Opin Struct Biol 23, 125-133, (2013). 23 Roca, J., Berger, J. M., Harrison, S. C. & Wang, J. C. DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism. Proc Natl Acad Sci U S A 93, 4057-4062 (1996). 24 Laponogov, I. et al. Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport. Nucleic Acids Res 41, 9911-9923, (2013). 25 Nollmann, M., Crisona, N. J. & Arimondo, P. B. Thirty years of Escherichia coli DNA gyrase: from in vivo function to single-molecule mechanism. Biochimie 89, 490-499, (2007). 26 Ullsperger, C. & Cozzarelli, N. R. Contrasting enzymatic activities of topoisomerase IV and DNA gyrase from Escherichia coli. J Biol Chem 271, 31549-31555 (1996). 27 Marians, K. J. DNA gyrase-catalyzed decatenation of multiply linked DNA dimers. J Biol Chem 262, 10362-10368 (1987). 28 McClendon, A. K., Rodriguez, A. C. & Osheroff, N. Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks. J Biol Chem 280, 39337-39345, (2005). 29 Wendorff, T. J., Schmidt, B. H., Heslop, P., Austin, C. A. & Berger, J. M. The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage. J Mol Biol 424, 109-124, (2012). 30 Bower, J. J. et al. Topoisomerase IIalpha maintains genomic stability through decatenation G(2) checkpoint signaling. Oncogene 29, 4787-4799, (2010). 31 Austin, C. A. & Marsh, K. L. Eukaryotic DNA topoisomerase II beta. Bioessays 20, 215-226, (1998). 32 Chen, S. H., Chan, N. L. & Hsieh, T. S. New mechanistic and functional insights into DNA topoisomerases. Annu Rev Biochem 82, 139-170, (2013). 33 Di Leo, A. et al. Topoisomerase II alpha as a marker predicting anthracyclines' activity in early breast cancer patients: ready for the primetime? Eur J Cancer 44, 2791-2798, (2008). 34 Nitiss, J. L. Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9, 338-350, (2009). 35 Larsen, A. K., Escargueil, A. E. & Skladanowski, A. Catalytic topoisomerase II inhibitors in cancer therapy. Pharmacol Ther 99, 167-181 (2003). 36 Chen, T., Sun, Y., Ji, P., Kopetz, S. & Zhang, W. Topoisomerase IIalpha in chromosome instability and personalized cancer therapy. Oncogene, (2014). 37 Pendleton, M., Lindsey, R. H., Jr., Felix, C. A., Grimwade, D. & Osheroff, N. Topoisomerase II and leukemia. Ann N Y Acad Sci 1310, 98-110, (2014). 38 Wasserman, R. A., Austin, C. A., Fisher, L. M. & Wang, J. C. Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast. Cancer Res 53, 3591-3596 (1993). 39 Wu, C. C. et al. Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide. Science 333, 459-462, (2011). 40 Russo Krauss, I., Merlino, A., Vergara, A. & Sica, F. An overview of biological macromolecule crystallization. Int J Mol Sci 14, 11643-11691, (2013). 41 Stanger, F. V., Dehio, C. & Schirmer, T. Structure of the N-terminal Gyrase B fragment in complex with ADPPi reveals rigid-body motion induced by ATP hydrolysis. PLoS One 9, e107289, (2014). 42 Dong, K. C. & Berger, J. M. Structural basis for gate-DNA recognition and bending by type IIA topoisomerases. Nature 450, 1201-1205, (2007). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52131 | - |
dc.description.abstract | 拓樸異構酶是生物體內不可或缺的酵素之一,不論原核或真核生物,都需要透過拓樸異構酶改變DNA拓樸構形的活性,移除細胞複製、轉錄及染色體重組等作用中產生的超螺旋、扭結或連鎖等阻礙反應進行的DNA結構,使DNA代謝作用得以順利完成。
拓樸異構酶依照其於反應過程中造成DNA斷裂數目的差異可分為兩大型:第I型酵素催化DNA單股斷裂而改變1個單位的DNA連結數 (Lk);第II型可使DNA雙股同時斷裂而讓Lk增減2個單位。每一型拓樸異構酶又依照序列相似性及催化機制再分為A、B兩個子類,而本篇論文研究的主題為屬於IIA型的人類拓樸異構酶IIα。目前對於IIA型拓樸異構酶的研究顯示,此類酵素的催化過程需要仰賴ATP,透過酵素活性中心的酪胺酸 (tyrosine) 對一段稱為G-segment的雙股DNA之磷酸雙酯鍵 (phosphodiester bond) 進行親核性攻擊形成磷酸酪胺基連接 (phosphotyrosyl linkage),並將此DNA切開而產生缺口,再利用與ATP的結合與水解促進另一段稱為T-segment的雙股DNA穿過此缺口,此過程涉及酵素四級結構大幅度的變化。然而目前對於反應不同階段所發生的構形變化尚需更多的結構資訊,才能對酵素的作用機制有更深入的認識。本研究中我們進行了近乎全長序列的人類拓樸異構酶IIα與DNA複合體的結構解析:首先使用酵母菌表現此蛋白,並且經液相層析與分子篩管柱進行純化,最後以活性試驗確認蛋白可形成結構正確並且具有正常催化活性的二聚體。 目前我們已經獲得人類拓樸異構酶IIα與DNA複合體的蛋白晶體,並且進行晶體培養條件的微調後,經由X-ray繞射數據及運算軟體得到解析度 (resolution) 約6.6 Å的蛋白結構。將此結構與先前已發表的酵母菌拓樸異構酶II (PDB:4GFH) 進行疊合比較,可發現我們解出的人類拓樸異構酶IIα之ATPase功能域 (domain) 與主要催化G-segment DNA結合及切割的活性區域 (DNA binding and cleavage core,DBCC) 相距較遠,猜測兩者應該分別對應於催化機制中不同的結構狀態。此外,由於我們的晶體結構可同時容納G-segment與T-segment DNA的存在,因此可能提供酵素藉由構形改變催化T-segment通過G-segment過程的重要資訊,所以我們設計了一段具有35-mer並且5’與3’分別模擬為G-segment及T-segment的DNA,希望未來能與人類拓樸異構酶IIα共同結晶,進而以X-ray繞射解得酵素同時與G-segment和T-segment產生交互作用的結構。 | zh_TW |
dc.description.abstract | Topoisomerases are essential enzymes ubiquitously present in eukaryotes, archaebacteria, and eubacteria. With their activities in manipulating DNA topology, these enzymes can resolve DNA entanglements and supercoils and are known to play critical roles in many cellular DNA transactions, including replication, transcription, recombination and chromatin remodeling.
Topoisomerases can be classified into two types based on whether one or both DNA strands of a duplex are cut during a catalytic event: type I and type II topoisomerases introduce single or double strand breaks, respectively, to alter the DNA linking number in steps of either one and two. Each type of topoisomerase can be further divided into two sub-families (A or B) depending on sequence similarities and differences in catalytic mechanisms. Higher eukaryotes encode 6 functionally distinct topoisomerases: Top1, Top1mt, Top2α, Top2β, Top3α, and Top3β. Our studies focus on the human topoisomerase IIα (hTop2α), which belongs to the type IIA category. Previous studies show that type IIA topoisomerases possess an ATP-dependent DNA passage activity. The enzyme binds to and cleaves one DNA duplex (termed the G-segment) via the formation of phosphotyrosyl linkages between a pair of catalytic tyrosines and DNA backbone phosphodiester bonds, thus opening up a gate on the G-segment for transporting another DNA duplex (termed the T-segment) through the break. The binding and hydrolysis of ATP are central for Top2α function, which drives protein conformational changes to allow the capture and transport of T-segment. However, structural basis underlying the transitions between the various conformational states of Top2α has remained unclear. To better understand the whole mechanism, we attempt to perform structural analysis on a near full length hTop2α in complex with DNA. The recombinant protein can be successfully expressed in yeast strain BCY123 and purified to homogeneity. Size exclusion chromatography revealed that hTop2α produced by yeast exists as dimers. Functional analysis further demonstrated that the recombinant hTop2α retains wild-type-like catalytic activities and sensitivity toward topoisomerase-targeting drugs. We have recently obtained crystals of the hTop2α-DNA binary complex that diffract to about 6.6 Å resolution. The diffraction quality of these crystals was optimized by refining the crystallization conditions to facilitate structural determination. Preliminary structural analysis shows that the ATPase domain and the DBCC (DNA binding and cleavage core) domain of hTop2α are further apart compared to the structure of yeast Top2 (PDB: 4GFH). Given that the T- and G-segment can be accommodated simultaneously in our structure, it appears that hTop2α may be trapped in a new conformational state during the enzymes catalytic cycle. Therefore, we designed a 35-mer DNA substrate, which mimics the presence of both the G-segment and the T-segment for co-crystallization with hTop2α. We expect to elucidate how hTop2α interacts with G-segment and T-segment simultaneously by performing X-ray diffraction analysis of hTop2α in complex with the 35-mer DNA. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:08:26Z (GMT). No. of bitstreams: 1 ntu-104-R02442009-1.pdf: 1845894 bytes, checksum: f6b8d1b659f2939b81172da498e96bee (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 謝誌 I
摘要 II Abstract IV 縮寫表 VII 目錄 VIII 圖目錄 XI 表目錄 XII 一、前言 1 1.1. DNA拓樸結構問題及拓樸異構酶 1 1.2. 拓樸異構酶之分類與功能 2 1.3. IIA型拓樸異構酶之結構與催化機制 4 1.4. 人類IIA型拓樸異構酶 6 1.5. 人類IIA型拓樸異構酶作為藥物治療標的 7 1.6. 研究動機 9 二、材料與方法 10 2.1. 蛋白質表現系統 10 2.1.1. 表現質體建構 10 2.1.2. 表現蛋白菌株 10 2.1.3. 製備酵母菌勝任細胞 (competent cell) 及轉型作用 11 2.1.4. hTop2α之大量表現 11 2.2. 蛋白質純化 11 2.2.1. 破菌與蛋白萃取 11 2.2.2. 液相層析 (liquid chromatography) 12 2.3. 蛋白質濃縮及定量 15 2.3.1. hTop2α濃縮 15 2.3.2. 定量hTop2α 15 2.4. 蛋白質活性測試 16 2.4.1. 電泳遷移率改變實驗 (electrophoresis mobility shift assay,EMSA) 16 2.4.2. hTop2α切割DNA試驗 (cleavage assay) 16 2.4.3. relaxation assay 17 2.5. 蛋白質晶體培養 17 2.5.1. 養晶樣品製備 17 2.5.2. 養晶前處理 18 2.5.3. 養晶方法 19 2.5.4. 養晶條件 20 2.6. 蛋白質晶體之X-ray繞射數據收集與結構解析 20 2.6.1. hTop2α晶體冷凍保護 (cryo-protection) 20 2.6.2. hTop2α晶體之X-ray繞射數據收集 21 2.6.3. hTop2α蛋白結構解析 21 2.7. 共養晶之DNA 22 2.7.1. 單股核苷酸序列設計 22 2.7.2. 雙股DNA形成 (duplex formation) 22 2.7.3. 限制酵素切割 22 2.7.4. 電泳遷移率改變實驗 (EMSA) 23 三、結果 24 3.1. 蛋白質純化 24 3.1.1. 離子交換層析 24 3.1.2. 膠體過濾層析 24 3.2. 蛋白質活性測試 24 3.2.1. 電泳遷移率改變實驗 (EMSA) 24 3.2.2. hTop2α切割DNA試驗 (cleavage assay) 25 3.2.3. relaxation assay 25 3.3. 蛋白質晶體培養 26 3.3.1. Natrix 1 No. 2 (Hampton) 26 3.3.2. 微晶種 (microseeding) 養晶 26 3.4. 蛋白質結構解析 26 3.4.1. X-ray繞射圖譜 26 3.4.2. hTop2α-DNA-VM-26結構 27 3.4.3. hTop2α與yeast Top2比較 27 3.5. DNA substrate設計 28 3.5.1. 雙股DNA形成 28 3.5.2. 限制酵素切割 28 3.5.3. 電泳遷移率改變實驗 (EMSA) 28 四、討論 29 4.1. 蛋白質表現 29 4.2. 蛋白質純化 29 4.3. 蛋白質晶體培養 30 4.4. DNA substrate設計 31 4.4.1. DNA序列設計 31 4.4.2. 雙股DNA製備 (duplex formation) 31 4.4.3. DNA雙股形成方式 32 4.4.4 G segment與T segment同時與hTop2α發生交互作用 32 圖 33 表 48 參考文獻 55 | |
dc.language.iso | zh-TW | |
dc.title | 人類第二型拓樸異構酶α亞型之結構解析 | zh_TW |
dc.title | Structural Analysis of Human DNA Topoisomerase IIα | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐駿森,曾秀如 | |
dc.subject.keyword | DNA拓樸結構,IIA型拓樸異構?,人類拓樸異構?IIα,晶體結構, | zh_TW |
dc.subject.keyword | DNA topology,Type IIA topoisomerases,human topoisomerase IIα,crystal structure, | en |
dc.relation.page | 57 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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