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研究生: 匡玉琪
Kuang, Yu-Chi
論文名稱: hRPA以不同方向性解開三核苷酸重複序列髮夾結構的機制研究
Polarity-dependent Mechanism of Human Replication Protein A (hRPA) Resolving Trinucleotide Repeat Hairpins
指導教授: 李以仁
Lee, I-Ren
口試委員: 冀宏源
Chi, Hung-Yuan
李弘文
Li, Hung-Wen
李以仁
Lee, I-Ren
口試日期: 2022/07/14
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 67
中文關鍵詞: 單分子螢光共振能量轉移三核苷酸重複序列CTG 重複序列單股DNA結合蛋白人類複製蛋白A方向性
英文關鍵詞: single-molecule fluorescence resonance energy transfer, trinucleotide repeat, CTG repeat, single-stranded binding protein, human replication protein A, polarity
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202201093
論文種類: 學術論文
相關次數: 點閱:73下載:0
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  • 三核苷酸重複序列的異常擴張容易引發許多神經退化性疾病,這些序列容易折疊成二級結構,導致 DNA 在複製、重組及修復時發生滑動,進而造成不正常擴張。人類複製蛋白 A (human replication protein A, hRPA) 是真核生物中含量最豐富的單股 DNA 結合蛋白,其功能在於幫助 DNA 保持在單股的狀態以維持基因組的穩定性,而其亦具有解開部分二級結構的能力並傾向從 5’ 端往 3’ 端解開。在本論文中,我們以 CTG 重複序列所形成的髮夾型結構作為模型系統,並利用單分子螢光共振能量轉移技術探討 hRPA 在不同方向性上對於 CTG 重複序列之解旋機制,首先我們發現人類複製蛋白 A 難以解開對齊髮夾型結構,因此我們將CTG 重複序列的髮夾結構末端,加上一段 10 個核苷酸的單股 DNA,形成突出型髮夾結構 (overhang hairpin),以利 hRPA 的初始結合,而結果顯示 hRPA 只能部分解開 CTG 重複次數較多的髮夾型結構。我們進一步發現當一顆 hRPA 結合 CTG 重複序列後,髮夾型結構會發生滑動重組,導致形成 hRPA 難以解開的對齊髮夾型結構。然而,在單點突變抑制滑動的實驗中,我們卻發現了與 DNA 方向性有關的結果:當 hRPA 從 3’ 端的突出 DNA 入侵時,單點突變會使其更容易形成完全解開結構;但當 hRPA 從 5’ 端的突出 DNA 入侵時,卻發現其結果有著更低比例的完全解開結構。因此,我們提出了一個模型:在hRPA 從 3’ 端往 5’ 端解開 CTG 髮夾型結構,第二顆 hRPA 便會結合上另一股以 5’ 端往 3’ 端的方向進行更進一步的入侵並解開整個髮夾型解構。然而,在另一個相反的方向性中,第二顆的 hRPA 則不傾向結合上另一股以 3’ 端往 5’ 端的方向解開 CTG 髮夾型結構,進而降低完全解開的效率。因此,我們可以總結在不同的方向性中,hRPA 具有不同的解旋機制。

    Abnormal expansions of trinucleotide repeats (TNRs) are responsible for many neurodegenerative disorders. TNRs usually fold into secondary structures that cause DNA slippage during DNA replication, recombination, or repair processes and ultimately lead to abnormal expansions. Human replication protein A (hRPA) is the most abundant single-stranded DNA binding protein in eukaryotes. Its major function is maintaining the single-stranded structure of DNA to keep genomic stability. It is also capable of resolving secondary structures with a polarity preference of 5’ to 3’. In the thesis, we used CTG repeat sequences, which fold into hairpins, as our model system to explore the mechanism of hRPA resolving CTG repeat hairpins in different polarities, utilizing single-molecule fluorescence resonance energy transfer (smFRET) microscopy. We found that hRPA cannot resolve the blunt-end hairpins. We then introduced a short (10-nt) random-coiled overhang to the hairpins for the initial binding of hRPA. The results revealed that hRPA partially resolves long hairpins. We further found that CTG repeat hairpin would undergo hairpin slippage and reorganize into blunt-end hairpin which prohibits the further invasion of hRPA. But when we introduced a single-point mutation to inhibit the slippage reconfiguration, we found polarity-dependent results: The point-mutation boosted the fully resolved hairpin when hRPA invaded from the overhang at 3’ end, while the lowered resolving efficiency was observed when hRPA invaded from the overhang at 5’ end. Hence, we proposed a model: After the hRPA resolves the CTG repeat hairpin from 3’ to 5’, the second hRPA binds to the other strand from 5’ to 3’ and further invades and fully resolves the hairpin. However, with the opposite polarity, the 3’ to 5’ invasion on the other stand is unfavorable and leads to a lowered resolving efficiency. Hence, we can conclude that hRPA has different resolving mechanisms in different polarities.

    摘要 i Abstract ii 目錄 iv 表目錄 vi 圖目錄 vii 第一章 、緒論 1 1.1 三核苷酸重複序列及其相關疾病 1 1.2 重複序列形成之非典型二級結構 3 1.3 重複序列的擴增機制 5 1.4 (CTG)n 重複序列之構型轉換 8 1.5 單股 DNA 結合蛋白 9 1.6 人類複製蛋白 A 11 1.7 hRPA 在不同方向性解開二級結構的能力 13 1.8 研究動機 15 第二章 、實驗儀器與方法 16 2.1 實驗儀器與原理 16 2.1.1 單分子實驗技術 16 2.1.2 螢光共振能量轉移 18 2.1.3 全內反射螢光顯微鏡 21 2.2 實驗方法與樣品製備 23 2.2.1 實驗樣品槽製備 23 2.2.2 DNA 序列設計 26 2.2.3 螢光分子的標記 28 2.2.4 DNA 黏合反應 29 2.2.5 分子間二聚體去除反應 30 2.2.6 固定實驗樣品於樣品槽 32 2.2.7 光閃爍與光漂白現象 33 2.2.8 顯影緩衝溶液 35 2.2.9 hRPA 與 eGFP-RPA 之真實濃度計算與儲存 37 2.3 儀器控制與數據分析 39 2.3.1 儀器控制 39 2.4 數據處理 41 2.4.1 數據動態擬合分析 43 第三章 、實驗結果與討論 44 3.1 單分子實驗設計 44 3.2 hRPA 與不同長度之 (CTG)nT10-ds 之結合 46 3.3 hRPA 與 (CTG)18T10-ds 結合之長時間軌跡圖 48 3.4 光漂白實驗 50 3.5 髮夾型結構滑動對hRPA結合的阻礙 51 3.6 改變螢光受體位置之實驗設計與結果 52 3.7 單點突變之實驗設計與結果 54 3.8 hRPA 對於不同方向性之三核苷酸重複序列的解旋能力 56 3.8.1 hRPA 解開不同方向性之髮夾型結構的差異 56 3.8.2 不同方向性之三核苷酸重複序列的滑動現象 58 3.8.3 hRPA對於三核苷酸重複序列之解旋機制 60 第四章 、結論與未來展望 63 4.1 結論 63 4.2 未來展望 65 參考資料 66

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