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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
dc.contributor.author | Han-Lin Yang | en |
dc.contributor.author | 楊翰霖 | zh_TW |
dc.date.accessioned | 2021-06-17T08:23:07Z | - |
dc.date.available | 2022-08-22 | |
dc.date.copyright | 2019-08-22 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-13 | |
dc.identifier.citation | 1 Windgassen, T. A., Wessel, S. R., Bhattacharyya, B. & Keck, J. L. Mechanisms of bacterial DNA replication restart. Nucleic Acids Res. 46, 504-519 (2017).
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74178 | - |
dc.description.abstract | 人類CTC1, STN1,及TEN1複合體 (CST) 蛋白和Replication Protein A (RPA) 皆為單股DNA結合蛋白,都具有與單股DNA結合的oligonucleotide / oligosaccharide binding (OB) 結構區域。我們使用高解析單分子螢光共振能量轉移 (smFRET),比較不同蛋白濃度下,CST、RPA與含多TG鹼基的3端單股DNA結合之FRET值分佈,量測結合、解離速率及解離常數。與RPA相比,CST在單股DNA上的解離常數較大,CST有數十秒尺度的結合/解離現象。隨著溶液中游離CST濃度增加,CST結合、脫離單股DNA速率皆會隨之上升。然而,當溶液中多餘的CST被移除時,CST與DNA的解離時間則超過1小時。表示CST的結合、解離速率和溶液中的CST濃度有關。RAD51是參與DNA同源重組修復的重要蛋白,RAD51會在單股DNA上形成核蛋白絲。在重啟停滯複製叉時,CST、RPA、RAD51等蛋白皆會參與反應。實驗發現當溶液加入RAD51同源重組酵素時,RAD51可以競爭並取代約30 % 原本結合在DNA上CST蛋白,形成RAD51核蛋白絲。然而,RAD51卻無法競爭已經結合在DNA上的RPA蛋白。儘管RPA比CST有更高的單股DNA親合力,當CST加入覆滿RPA的單股DNA,CST依然可結合上覆滿RPA的單股DNA。實驗結果指出CST在單股DNA上的結合、解離和OB區域結構與CST部分解離有關,露出部分的單股DNA,讓RAD51有機會在該段單股DNA上成核,形成核蛋白絲。 | zh_TW |
dc.description.abstract | Human CST (CTC1, STN1, and TEN1) complex and Replication Protein A (RPA) are single-stranded DNA (ssDNA) binding proteins. Both contain several canonical OB-fold domains responsible for ssDNA binding. We used high-resolution single-molecule fluorescence resonance energy transfer (smFRET) experiments to directly measure the association and dissociation of CST and RPA on TG-rich 3′ overhang ssDNA, and determined the thermodynamics and kinetics parameters. RPA stably stays bound to DNA, but CST binds and dissociates dynamically in tens of seconds. Interestingly, at higher CST concentrations, both their on rate and off rate increase. However, the dissociation dwell time of CST becomes very long (> 1 hour) when the solution contains no free CST, suggesting that CST dissociation depends on the excess CST in the solution. RAD51 is an essential protein involved in DNA homologous recombination. RAD51 can oligomerize on the ssDNA to form nucleoprotein filaments. CST, RPA, and RAD51 are important components in rescuing stalled replication forks. RAD51 can remove roughly 30 % CST from the CST-bound ssDNA and form stable RAD51 nucleoprotein filament. However, RAD51 alone cannot remove the RPA from RPA-bound ssDNA. Despite the fact that RPA has stronger ssDNA binding affinity than CST, CST can still bind to the RPA-bound ssDNA. In summary, our model suggests that partial dissociation of OB-fold domains of CST is responsible for the dynamic nature of CST binding. Thus, RAD51 can nucleate on the exposed ssDNA originally occupied by CST to assemble nucleoprotein filament. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:23:07Z (GMT). No. of bitstreams: 1 ntu-108-R06223129-1.pdf: 6626264 bytes, checksum: 8bb448b5cd027bcc991c3f21efe8d8dd (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 圖目錄 iv
表目錄 vi 1 緒論 1 1.1 重啟複製叉 1 1.2 RAD51 3 1.3 RPA 5 1.4 CST 6 1.5 研究動機 8 2研究方法與步驟 9 2.1 蛋白質來源 9 2.2 DNA基質設計 9 2.3 螢光顯微鏡架設 11 2.4 PEG修飾玻片製作 14 2.5 PCR DNA黏合過程 17 2.6 溶液除氧系統配方 18 2.7 實驗流程 19 2.8 單分子螢光共振能量轉移數據處理 26 3 實驗結果與討論 36 3.1 CST結合TG-rich 15 nt解離常數為1.5 nM 36 3.2 RPA結合DNA能力比CST好 39 3.3 在短單股DNA下,CST偏好G-rich序列 40 3.4 FRET軌跡顯示CST的Rateon & Rateoff皆隨[CST]上升而增加 42 3.5 溶液中沒有游離CST時,CST脫落DNA停滯時間約2小時 46 3.6 Non-G rich下,RAD51的FRET值和RPA、CST都不同 48 3.7 RAD51可競爭已結合單股DNA的CST,形成filament 50 3.8 CST可結合覆滿RPA的單股DNA 56 4 結論與未來展望 60 4.1 結論 60 4.2 未來展望 62 螢光修飾CST、RPA 62 螢光光鉗 63 純化RPA、CST的單一組成蛋白或缺少一組成的雙異核蛋白 63 參考文獻 65 | |
dc.language.iso | zh-TW | |
dc.title | 利用單分子螢光共振能量轉移研究人類端粒結合蛋白的熱力學和動力學性質 | zh_TW |
dc.title | Single Molecule FRET Identified the Dynamic Nature of Human Telomere-Binding Protein CST Complex | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 冀宏源(Hung-Yuan (Peter),林敬哲(Jing-Jer Lin),詹迺立(Nei-Li Chan),范秀芳(Hsiu-Fang Fan) | |
dc.subject.keyword | 單分子螢光共振能量轉移,CST,RAD51,RPA,停滯複製叉, | zh_TW |
dc.subject.keyword | smFRET,CST,RAD51,RPA,stalled replication fork, | en |
dc.relation.page | 67 | |
dc.identifier.doi | 10.6342/NTU201903225 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-13 | |
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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