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Title: | 利用單分子螢光技術探討人類RAD51蛋白與端粒RNA的相互作用 Study the interaction between human RAD51 and telomeric RNA by single molecule fluorescence techniques |
Authors: | 梁宗仁 Chong-Ren Neoh |
Advisor: | 李弘文 Hung-Wen Li |
Keyword: | 人類端粒,端粒RNA,G-四聯體,RAD51,單分子螢光共振能量轉移,單分子螢光顯微鏡, Telomere,TERRA,G-quadruplex,RAD51,Single molecule FRET,Single molecule fluorescence microscopy, |
Publication Year : | 2022 |
Degree: | 碩士 |
Abstract: | 人類的端粒 (Telomere) 不僅可以保護染色體的末端,其序列的長度也與細胞老化息息相關。在細胞中有不少蛋白可與 Telomere 作用並形成特殊結構,但當其 以單股的形式裸露時也會形成特殊的 G-quadruplex (G4) 二級結構。雖然目前已知 細胞可以通過端粒酶 (Telomerase) 來延長端粒的長度,避開由DNA複製所造成的 端粒縮短問題,但有文獻指出在不具有端粒酶表達活性的細胞株中發現端粒也有 被延長的現象,更有不少的文獻指出端粒 RNA (TERRA) 有可能參與其中,且有 可能是通過人類 RAD51 蛋白 (hRAD51) 協助完成端粒的延長。我們奠基在前人的 研究成果上,進一步以單分子螢光共振能量轉移 (smFRET) 與單分子螢光共位實 驗來探討 hRAD51 與 Telomere 以及TERRA的相互作用。我們發現,G4 結構可以 很輕易的在 Telomere 以及 TERRA 上形成,且 hRAD51 非常具有特異性的只結合 到 TERRA 的 G4 上而不結合到 Telomere 的 G4 上,藉此造成了親和力上的差別。 特別的是,hRAD51 雖然可以結合到 TERRA G4 上,但卻不是以延伸型核蛋白絲 的方式結合。進一步分析我們的實驗數據,我們發現約有 6 至 7 個 hRAD51 可以 結合到 TERRA G4 上。最後,我們的實驗結果說明,這些穩定結合在 TERRA G4 上的 hRAD51 並不具有捕捉雙股DNA的能力。 Human telomere not only protects the chromosome’s end, but its length also related to cell senescence. When telomere is exposed in single-stranded form, it forms into a special secondary structure, G-quadruplex (G4). Although telomere lengthening through telomerase has been well known, recent studies observed telomere lengthening events also occur in telomerase deletion cells. Some studies have suggested that TERRA might be involved in telomere lengthening possibly through human RAD51 protein (hRAD51). Our study base on previous results and steps one step further by using single-molecule techniques to study the interaction between telomere, TERRA, and hRAD51. Our results showed that G4 structure is readily formed on both telomere and TERRA. Surprisingly, we found that hRAD51 specifically bind to TERRA G4 but not telomere G4 without forming extended nucleoprotein filament. Through analysis, we assume there is about 6 to 7 hRAD51 bind to single TERRA G4. Lastly, our results showed that hRAD51 that bound on TERRA G4 didn’t have effective duplex capture ability. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87563 |
DOI: | 10.6342/NTU202300495 |
Fulltext Rights: | 同意授權(限校園內公開) |
metadata.dc.date.embargo-lift: | 2028-02-18 |
Appears in Collections: | 化學系 |
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ntu-111-1.pdf Restricted Access | 13.35 MB | Adobe PDF | View/Open |
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