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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃婉媜(Wan-Chen Huang) | |
dc.contributor.author | Lin-Ying Wu | en |
dc.contributor.author | 吳琳瑩 | zh_TW |
dc.date.accessioned | 2021-06-08T03:26:35Z | - |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-03-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21065 | - |
dc.description.abstract | DNA拓樸異構酶(DNA Topoisomerase,Top)是一種可藉由其切割-再接合活性解決DNA拓樸結構問題(DNA topological problem)的酵素。依據作用機轉的不同,拓樸異構酶可分為兩種類型:第一型拓樸異構酶(Type I topoisomerase)與第二型拓樸異構酶(Type II topoisomerase)。第一型拓樸異構酶不需仰賴三磷酸腺苷(Adenosine Triphosphate,ATP)供給能量即可使DNA單股暫時斷裂,第二型拓樸異構酶則需要ATP參與供給能量才能使DNA雙股暫時斷裂。兩種類型的拓樸異構酶皆是藉由活性中心上的酪胺酸(tyrosine)打斷DNA的磷酸雙酯鍵(phosphodiester bond),進而使DNA產生暫時性的斷裂。依據胺基酸序列及結構的相似性,兩種類型的拓樸異構酶又可更進一步的細分為A及B兩種亞型(subfamily)。本篇論文的研究主題為探討Type IIA中人類第二型拓樸異構酶α (human Topoisomerase II α,hTop2α)與雙股DNA相互作用間各個反應步驟的動態變化。 目前已可藉由單分子技術(single-molecule techniques)與螢光共振能量轉移技術(Fluorescence Resonance Energy Transfer,FRET)進行DNA-binding protein彎曲DNA結構的動態分析。本論文即是藉由兩項技術的結合,搭配物鏡型全內反射式螢光顯微鏡(Total Internal Reflection Fluorescence Microscopy,TIRFM)及高靈敏性的偵測系統,即時觀測hTop2α與雙股DNA相互作用間各個反應步驟的動態變化。 本論文的研究結果顯示本實驗室自行純化的hTop2α的確可彎曲雙股DNA。外加ATP後,high EFRET DNA彎曲群體的增加及雙股DNA處於彎曲構型時間的延長皆表明ATP的存在可能讓DNA更傾向於彎曲的構型。此外,使用relaxation assay測試藥物或化合物抑制hTop2α活性的結果中則發現化合物0785(濃度約200 nM時幾乎可完全抑制hTop2α活性)較目前臨床常用的hTop2α抑制劑-dexrazoxane (ICRF–187)(濃度達600 nM時亦未完全抑制hTop2α活性)更可顯著抑制hTop2α。未來期望藉由單分子螢光共振能量轉移(single-molecule Fluorescence Resonance Energy Transfer,smFRET)影像技術結合高靈敏性的TIRFM偵測系統尋找出化合物0785及其他化合物或藥物對hTop2α可能的作用干擾機制。 | zh_TW |
dc.description.abstract | DNA topoisomerases (Top) are enzymes that resolve DNA topological problems via their DNA cleavage-religation activity. Based on different DNA cleavage mode, single-stranded or double-stranded DNA breaks, topoisomerases are classified into type I and type II, respectively. Both types of topoisomerases break phosphodiesterase bond of DNA nucleotides through the tyrosine residue in the active site, introducing temporary breaks in DNA. The aim of this study is to investigate the dynamic changes of the interactions, such as DNA bending after protein binding, DNA opening and closing after DNA cleavage, between human topoisomerase II α (hTop2α) and double-stranded DNA. Currently, the dynamic analysis of DNA bending by DNA-binding protein can be observed by using single-molecule techniques combined with fluorescence resonance energy transfer (smFRET). Here, we further applied smFRET approach based on objective-based total internal reflection fluorescence microscopy (TIRFM) with highly sensitive detection system to observe the real-time dynamic changes of interactions between hTop2α and double-strand DNA at each catalytic step. Our results showed that the hTop2α, which is generated in our laboratory, can induce double-strand DNA bending. Additionally, both the probability and dwell time of high EFRET, representing DNA bending, are increase after ATP treatment indicating that DNA tended to form a bent conformation in the presence of hTop2α and ATP. Moreover, comparing with dexrazoxane (ICRF-187), a known clinically used hTop2α inhibitor, with compound 0785, the latter exhibited a better inhibitory effect on hTop2α relaxation activity. Hopefully, we could apply the smFRET technique based on TIRF microscopy to explore the possible inhibitory mechanisms of novel compound 0785 on the hTop2α-DNA interaction. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:26:35Z (GMT). No. of bitstreams: 1 U0001-2503202118344400.pdf: 6119634 bytes, checksum: 3a2731a1834c67617ce1cb7e00a7cc6b (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 口試委員會審定書 I 致謝 II 中文摘要 III Abstract V 目錄 VII 圖目錄 X 表目錄 XII 縮寫表 XIII 第一章 緒論 1 1.1文獻回顧 1 1.1.1 DNA拓樸結構與拓樸異構酶 1 1.1.2拓樸異構酶的分類 2 1.1.3真核生物Top2的結構及其作用機制 3 1.1.4人類第二型拓樸異構酶α 5 1.1.5人類拓樸異構酶作為藥物治療標的 6 1.2單分子螢光共振能量轉移影像技術 7 1.2.1單分子技術 8 1.2.2螢光共振能量轉移 8 1.2.3全內反射式螢光顯微鏡 10 1.3研究動機 10 1.4論文大綱 11 第二章 材料與方法 12 2.1 DNA序列的黏合與純化 12 2.2蛋白質表現系統 12 2.3蛋白質抽取與純化 13 2.3.1養菌、破菌與蛋白質抽取 13 2.3.2鎳離子管柱層析 14 2.3.3 SP管柱層析 14 2.3.4 MONO-S管柱層析 15 2.3.5 HiTrap-heparin管柱層析 15 2.3.6蛋白質透析 16 2.4蛋白質定量-coomassie blue staining 17 2.5 Relaxation assay 17 2.6單分子螢光共振能量轉移影像技術實驗儀器的架設 18 2.7實驗樣品製備 19 2.7.1玻片清理與修飾 19 2.7.2樣品槽組裝 19 2.7.3固定樣品於樣品槽 20 2.7.4影像緩衝溶液 20 2.8數據處理與分析 21 第三章 結果與討論 24 3.1雙股DNA的黏合與純化 24 3.2 hTop2α蛋白的純化 24 3.2.1以鎳離子管柱層析純化hTop2α蛋白 25 3.2.2以SP管柱層析純化hTop2α蛋白 25 3.2.3以MONO-S管柱層析純化hTop2α蛋白 26 3.2.4以heparin管柱層析純化hTop2α蛋白 26 3.2.5 hTop2α蛋白的定量 27 3.2.6 hTop2α蛋白的relaxation assay 27 3.2.7兩批hTop2α蛋白純化結果的比較 28 3.3單分子螢光共振能量轉移影像技術 28 3.3.1實驗玻片的清理與修飾 28 3.3.2 樣品DNA濃度的選擇 29 3.3.3 螢光共振能量轉移效率的校正 29 3.3.4 hTop2α蛋白與DNA間的作用 30 3.4化合物0785抑制hTop2α蛋白活性 31 第四章 結論 33 圖 34 表 68 參考文獻 69 | |
dc.language.iso | zh-TW | |
dc.title | 利用單分子螢光共振能量轉移影像技術觀察人類第二型拓樸異構酶α與DNA交互作用之動態變化 | zh_TW |
dc.title | Single-molecule Fluorescence Resonance Energy Transfer Imaging Technology Discloses the Dynamic Analysis of DNA and Human Topoisomerase 2α | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 吳文超(Wen-Chau Wu) | |
dc.contributor.oralexamcommittee | 張宜仁(Yi-Ren Chang) | |
dc.subject.keyword | 人類第二型拓樸異構酶α,單分子技術,螢光共振能量轉移技術,全內反射式螢光顯微鏡, | zh_TW |
dc.subject.keyword | Human Topoisomerase II α,Single-molecule Technique,Fluorescence Resonance Energy Transfer,Total Internal Reflection Fluorescence Microscopy, | en |
dc.relation.page | 80 | |
dc.identifier.doi | 10.6342/NTU202100805 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-04-07 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫療器材與醫學影像研究所 | zh_TW |
顯示於系所單位: | 醫療器材與醫學影像研究所 |
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