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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
dc.contributor.author | Yu-Hsuan Lin | en |
dc.contributor.author | 林宇軒 | zh_TW |
dc.date.accessioned | 2021-06-17T01:26:17Z | - |
dc.date.available | 2019-08-25 | |
dc.date.copyright | 2017-08-25 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-07 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67279 | - |
dc.description.abstract | RAD51 催化的同源重組修復反應是真核生物體內很重要的 DNA 修補機制,在含有 ATP 的情況下, RAD51 可與單股 DNA 結合並形成核蛋白絲,此核蛋白絲會啟動同源重組修復反應,以修復致命的 DNA 雙股斷裂。 SWI5-SFR1 複合體已被報導具有穩定 RAD51 核蛋白絲的功能,但其穩定核蛋白絲的機制尚未明瞭。本研究強化並提高現有的單分子光鉗技術的穩定度,包括引入 PID 控制器進行雷射強度控制、使用能量譜密度分析進行雜訊偵測,並在實驗過程中同步進行定力鉗實驗操作與影像擷取,以利事後影像處理扣除雜訊,成功達到 1 nm 解析度。並利用此技術試圖研究老鼠種 RAD51 形成核蛋白絲的過程,包括與 DNA 結合以及自 DNA 上脫離的動力學性質,如何受到 SWI5-SFR1 複合體的調控。實驗發現在含有 ATP 的情況下, SWI5-SFR1 可提高 mRAD51 與雙股 DNA 結合的速率 kext,但並不影響 mRAD51 自雙股 DNA 上脫離的速率 kdis。另一方面,SWI5-SFR1 可提高 mRAD51 與單股 DNA 結合的速率 kext,同時抑制 mRAD51 自單股 DNA 上脫離的反應 kdis。此調控功能暗示 SWI5-SFR1 不只可以有效的穩定 mRAD51 核蛋白絲,亦有可能使 mRAD51 在股交換反應完成後,迅速地脫離 DNA,使其能夠進行下一步的修復反應。 | zh_TW |
dc.description.abstract | Homologous recombination catalyzed by RAD51 recombinases is a crucial DNA repair pathway in eukaryotes. In the presence of ATP, RAD51 assembles on single-stranded DNA to form nucleoprotein filaments, and initiates homologous recombinational repair of DNA double-stranded breaks. The SWI5-SFR1 complex has been found to regulate RAD51 filament assembly and enhance strand exchange activity, but the detailed mechanism is not clear. Here we improved our home-built optical tweezers platform to 1 nm resolution, and utilized it to study the assembly and disassembly dynamics of mRAD51 filaments in the presence of SWI5-SFR1 complex. In the case of double-stranded DNA, mRAD51 assembly process is stimulated in the prescence of SWI5-SFR1, but the disassembly process is not affected. On the other hand, mRAD51 assembles onto single-stranded DNA with an enhanced rate in the prescence of SWI5-SFR1, and the disassembly process from ssDNA is suppressed by the SWI5-SFR1 complex. This indicates that the SWI5-SFR1 stabilization function happens in the mRAD51 nucleoprotein filament formation onto single-stranded DNA, while SWI5-SFR1 only alters the kext of double-stranded DNA filament formation, not kdis. These regulatory functions of SWI5-SFR1 imply not only efficient stabilization of mRAD51 nucleoprotein filament during strand exchange, but also offer efficient mRAD51 turnover once the reaction is completed. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T01:26:17Z (GMT). No. of bitstreams: 1 ntu-106-R03223147-1.pdf: 4474383 bytes, checksum: b4e2d3d11604d4724e46d2e7fd210f68 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii 目錄 iv 圖目錄 vii 表目錄 ix 第1章 緒論 1 1.1 同源重組反應 (Homologous Recombination) 1 1.2 反應機制 1 1.2.1 RAD51核蛋白絲形成步驟 3 1.2.2 輔助蛋白SWI5-SFR1對RAD51的影響 5 1.2.2.1 SWI5-SFR1促進mRAD51股交換反應活性 6 1.2.2.2 SWI5-SFR1穩定mRAD51核蛋白絲 6 1.2.2.3 複合體以 SWI5 C端與 mRAD51 核蛋白絲交互作用 8 1.3 研究動機 8 第2章 實驗方法與設計 10 2.1 RAD51與SWI5-SFR1蛋白的純化與保存 10 2.2 DNA 基質合成 10 2.2.1 全雙股 DNA 合成 10 2.2.2 無二級結構全單股 DNA 合成 11 2.2.3 Gap DNA 合成 15 2.3 實驗操作方法 18 2.3.1 製備光鉗實驗用 DNA 拴球 18 2.3.2 除氧系統 19 2.3.3 反應緩衝液 20 第3章 光鉗系統架設 21 3.1 光鉗平台原理及架設 21 3.1.1 光鉗基礎原理 21 3.1.2 光鉗系統設計概念 22 3.1.3 設定參考光路與檢驗光路平行 26 3.1.4 透鏡基本原理與光束縮放 27 3.1.5 光路調校 30 3.1.6 聲光調變器 (AOM/AOD) 32 3.1.7 光學系統架設步驟 33 3.1.8 自動化光鉗程式系統 34 3.2 建立PID控制雷射強度回饋系統 35 3.3 位置校正 37 3.3.1 決定雷射中心距離表面高度 37 3.3.2 建立QPD訊號與球相對雷射中心位置之校正線 38 3.4 測量光鉗力常數 38 3.4.1 能量密度譜分析法 (Power Spectrum Analysis Method) 39 3.5 量測 DNA 分子之長度與硬度資訊 (Force-Extension Assay) 41 3.6 定力鉗實驗即時偵測 DNA 分子之長度變化 (Force Clamp Assay) 42 3.7 玻片漂移之後期影像處理 45 3.7.1 計算玻片高度漂移 45 3.7.2 計算玻片側向漂移 47 第4章 實驗結果與討論 48 4.1 即時偵測 SWI5-SFR1 穩定 mRAD51 核蛋白絲 48 4.2 SWI5-SFR1 提高 mRAD51 對 dsDNA 的kext,但不影響 kdis 51 4.3 SWI5-SFR1 提高 mRAD51 對 ssDNA 的kext,亦降低 kdis 52 4.4 SWI5-SFR1 以穩定核蛋白絲之方式影響 mRAD51 股交換反應模型 53 第5章 結論與未來展望 56 5.1 結論 56 5.2 未來展望 57 參考文獻 62 附錄 70 附錄一 試劑與耗材清單 70 附錄二 DNA 引子序列 72 附錄三 單分子拴球玻片製備方法 73 附錄四 表面鏈霉親合素修飾之聚苯乙烯球製備步驟 (整鍋反應法) 74 附錄五 Pyranose Oxidase / Catalase (POC) 除氧系統的製備 75 附錄六 以能量均分定理 (Equipartition Theorem) 量測光鉗力常數 76 附錄七 光鉗 LabVIEW 程式說明文件 77 | |
dc.language.iso | zh-TW | |
dc.title | 以單分子光鉗系統探討SWI5-SFR1調控老鼠RAD51核蛋白絲形成動力學之研究 | zh_TW |
dc.title | Investigating How Mouse RAD51 Filament Dynamics Regulated by SWI5-SFR1 Complex Using Optical Tweezers | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王廷方(Ting-Fang Wang),冀宏源(Hung-Yuan (Peter),溫進德(Jin-Der Wen),李以仁(I-Ren Lee) | |
dc.subject.keyword | 同源重組,DNA 修復,RAD51,SWI5-SFR1,單分子生物物理,光鉗, | zh_TW |
dc.subject.keyword | homologous recombination,DNA repair,RAD51,SWI5-SFR1,single molecule biophysics,optical tweezers, | en |
dc.relation.page | 83 | |
dc.identifier.doi | 10.6342/NTU201602155 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-08 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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