- 學位論文
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
Single-molecule Fluorescence Resonance Energy Transfer Imaging Technology Discloses the Dynamic Analysis of DNA and Human Topoisomerase 2α
摘要
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。
並列摘要
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.
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