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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲(Jing-Jer Lin) | |
| dc.contributor.author | Yu-Ting Lin | en |
| dc.contributor.author | 林郁庭 | zh_TW |
| dc.date.accessioned | 2022-11-25T07:49:04Z | - |
| dc.date.available | 2023-10-01 | |
| dc.date.copyright | 2021-11-08 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-29 | |
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(2000). Identification of the single-strand telomeric DNA binding domain of the Saccharomyces cerevisiae Cdc13 protein. Proceedings of the National Academy of Sciences 97, 6457-6462. 12. Ishikawa-Ankerhold, H.C., Ankerhold, R., and Drummen, G.P. (2012). Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17, 4047-4132. 13. Jackson, C. (2011). Multi-state models for panel data: the msm package for R. Journal of statistical software 38, 1-28. 14. Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N., and Mann, M. (2014). Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319-324. 15. Larrivée, M., LeBel, C., and Wellinger, R.J. (2004). The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex. Genes development 18, 1391-1396. 16. Lendvay, T.S., Morris, D.K., Sah, J., Balasubramanian, B., and Lundblad, V. (1996). 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The telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures. PLoS Genet 9, e1003145. 26. Martín, V., Du, L.-L., Rozenzhak, S., and Russell, P. (2007). Protection of telomeres by a conserved Stn1–Ten1 complex. Proceedings of the National Academy of Sciences 104, 14038-14043. 27. McClintock, B. (1939). The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proceedings of the National Academy of Sciences of the United States of America 25, 405. 28. McClintock, B. (1941). The stability of broken ends of chromosomes in Zea mays. Genetics 26, 234. 29. Meyne, J., Ratliff, R.L., and MoYzIs, R.K. (1989). Conservation of the human telomere sequence (TTAGGG) n among vertebrates. Proceedings of the National Academy of Sciences 86, 7049-7053. 30. Mitchell, M.T., Smith, J.S., Mason, M., Harper, S., Speicher, D.W., Johnson, F.B., and Skordalakes, E. (2010). 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TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266, 404-409. 42. Sun, J., Yang, Y., Wan, K., Mao, N., Yu, T.Y., Lin, Y.C., DeZwaan, D.C., Freeman, B.C., Lin, J.J., Lue, N.F., et al. (2011). Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase alpha. Cell Res 21, 258-274. 43. Wang, M.-J., Lin, Y.-C., Pang, T.-L., Lee, J.-M., Chou, C.-C., and Lin, J.-J. (2000). Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p. Nucleic acids research 28, 4733-4741. 44. Wang, S., and Zakian, V.A. (1990). Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains. Molecular and cellular biology 10, 4415. 45. Wu, Y., and Zakian, V.A. (2011). The telomeric Cdc13 protein interacts directly with the telomerase subunit Est1 to bring it to telomeric DNA ends in vitro. Proceedings of the National Academy of Sciences 108, 20362-20369. 46. Zappulla, D.C., Roberts, J.N., Goodrich, K.J., Cech, T.R., and Wuttke, D.S. (2009). Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic acids research 37, 354-367. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82670 | - |
| dc.description.abstract | 端粒在維持真核生物染色體完整性上扮演重要角色。Cdc13為單股TG1–3端粒DNA結合蛋白，在Saccharomyces cerevisiae中主要以二聚體形式存在，此外，Cdc13與Stn1、Ten1形成CST複合物並結合在端粒末端，可保護端粒DNA序列及調控端粒酶之活性；在傳統生化分析與蛋白結構分析中均顯示Cdc13可特異性地與端粒DNA進行交互作用，然而與端粒穩定結合之Cdc13實際上是如何坐落至端粒DNA上，並和其他蛋白進行「動態」交互作用的，直至今日都還尚未明瞭，因此我們利用單分子螢光共振能量轉移(smFRET)實驗，以觀察Cdc13和端粒DNA間之交互作用。結果顯示，Cdc13與TG12單尾雙股DNA受質結合後，會降低DNA受質之螢光能量轉移效率至兩個不同的數值，即兩種FRET state。除此之外，進一步利用Cdc13-dimer mutant與DNA-binding domain (DBD) mutant蛋白進行實驗，可得知此兩種FRET state分別為Cdc13 monomer和dimer結合至端粒DNA上所造成，且利用即時影像實驗進行動力學分析可發現monomer結合速率較dimer結合速率快。依據實驗結果可建立一初步之Cdc13結合端粒DNA結合模型， Cdc13 以monomer形式優先結合至單股端粒DNA，第二個monomer則接續加入並形成dimer。為了區分Cdc13 monomer和dimer結合模式是否具有相異的生物功能，我們進一步使用smFRET觀察Cdc13與Stn1或Cdc13、Stn1與Ten1間之交互作用。結果表明，Stn1並不會影響Cdc13結合端粒DNA能力，然而CST複合物可能輕微減弱Cdc13和DNA間之親和力。綜合上述，本篇研究透過一單分子實驗建立了一個較為詳細的Cdc13結合酵母菌端粒DNA模式。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T07:49:04Z (GMT). No. of bitstreams: 1 U0001-2809202111234600.pdf: 5340445 bytes, checksum: 51f9e387a348a7532a76f74c3fb8c29b (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "謝辭 i 中文摘要 iv Abstract v 目錄 vi 前言 1 材料與方法 7 結果 43 討論 60 參考文獻 64 附表 69 表一、引子列表 69 表二、建構pFastBac1-FLAG-CDC13-R635C質體所使用之PCR配方 70 表三、建構pFastBac1-FLAG-CDC13-R635C質體之Gibson assembly配方 71 表四、同位素電泳膠遲緩分析之反應條件 72 表五、螢光電泳膠遲緩分析之反應條件(5% polyacrylamide gel) 73 表六、螢光電泳膠遲緩分析之反應條件(1% agarose gel) 74 表七、合成螢光標記DNA受質之配方 75 表八、Trolox配方 76 表九、酵素除氧系統配方 77 表十、Imaging buffer配方 78 表十一、EM-CCD之參數 79 表十二、平衡態實驗各個state所佔比例之原始數據 80 表十三、Hypothesis 1之dwell time及反應速率分析數據(Cdc13-WT) 81 表十四、Hypothesis 1中state 1到3之dwell time預測(Cdc13-WT) 82 表十五、Hypothesis 2之dwell time及反應速率分析數據(Cdc13-WT) 83 表十六、Hypothesis 2之dwell time及反應速率分析數據(Cdc13-dimer mutant) 84 表十七、Cdc13 heterodimer之biexponential decay fitting分析結果 85 表十八、Hypothesis 2之dwell time及反應速率分析數據(Cdc13 heterodimer) 86 表十九、Cdc13, Stn1, Ten1平衡態實驗各state比例之T-test檢定結果 87 附圖 88 圖一、設計帶有酵母菌端粒序列之螢光標記DNA受質 88 圖二、純化Cdc13相關重組蛋白之純度測試 89 圖三、純化Cdc13相關重組蛋白之活性測試 90 圖四、Cdc13與TG12 end DNA結合產生兩個較低的FRET state 91 圖五、State 3生成需要Cdc13形成dimer 92 圖六、即時影像實驗可觀察Cdc13與TG12 end DNA結合之動態變化 93 圖七、Cdc13-WT和Cdc13-dimer mutant即時影像實驗之時間軌跡圖 94 圖八、Cdc13與端粒DNA結合模型(Hypothesis 1) 95 圖九、利用指數衰減擬合(exponential decay fitting)進行dwell time分析 96 圖十、Cdc13-WT即時影像實驗之dwell time及反應速率分析(Hypothesis 1) 97 圖十一、Cdc13與端粒DNA結合模型(Hypothesis 2) 98 圖十二、Cdc13-WT即時影像實驗之dwell time及反應速率分析 99 圖十三、Cdc13-dimer mutant即時影像實驗之dwell time及反應速率分析 100 圖十四、Cdc13-R635C造成蛋白與DNA結合能力缺失 101 圖十五、State 3的產生需要Cdc13第二個DNA binding domain 102 圖十六、Cdc13 heterodimer即時影像實驗之dwell time及反應速率分析 103 圖十七、建立Cdc13與端粒DNA結合模型 104 圖十八、Cdc13-DBD無法產生state 3 105 圖十九、Stn1不會影響Cdc13與TG12 end DNA結合 106 圖二十、CST complex輕微降低Cdc13與TG12 end DNA結合之親和力 107 附錄 108 附錄一、Cdc13於細胞週期各階端中調控端粒DNA長度之機制 108 附錄二、pBac6H-Cdc13基因圖譜 109 附錄三、pET6H-CDC13-BD基因圖譜 110 附錄四、pFastBac1-FLAG-CDC13-R635C基因圖譜 111 附錄五、pRS424-STN1-TAP基因圖譜 112 附錄六、Cdc13 heterodimer重組蛋白純化 113" | |
| dc.language.iso | zh-TW | |
| dc.title | 利用單分子螢光共振能量轉移實驗分析Cdc13與端粒結合之模式 | zh_TW |
| dc.title | Single-molecule fluorescence resonance energy transfer (smFRET) analysis of telomeric DNA binding by Cdc13 protein | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李弘文(Hsin-Tsai Liu),詹迺立(Chih-Yang Tseng) | |
| dc.subject.keyword | 端粒,Cdc13,Stn1,Ten1,CST complex,單分子螢光共振能量轉移, | zh_TW |
| dc.subject.keyword | Telomere,Cdc13,Stn1,Ten1,CST complex,Single-molecule fluorescence resonance energy transfer, | en |
| dc.relation.page | 113 | |
| dc.identifier.doi | 10.6342/NTU202103423 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-09-30 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-10-01 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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