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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Ting-Wei Liao | en |
| dc.contributor.author | 廖庭尉 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:45:29Z | - |
| dc.date.available | 2020-08-02 | |
| dc.date.copyright | 2018-08-02 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-02 | |
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Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739-750 (2006). 8 Bugreev, D. V. Mazin, A. V. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl Acad. Sci. U S A 101, 9988-9993 (2004). 9 Ristic, D. et al. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res. 33, 3292-3302 (2005). 10 Chi, P., Van Komen, S., Sehorn, M. G., Sigurdsson, S. Sung, P. Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst). 5, 381-391 (2006). 11 Hilario, J., Amitani, I., Baskin, R. J. Kowalczykowski, S. C. Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl Acad. Sci. U S A 106, 361-368 (2009). 12 van Mameren, J. et al. Counting RAD51 proteins disassembling from nucleoprotein filaments under tension. Nature 457, 745-748 (2009). 13 Candelli, A. et al. Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution. Proc. Natl Acad. Sci. U S A 111, 15090-15095 (2014). 14 Moerner, W. E. Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535-2538 (1989). 15 Orrit, M. Bernard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716-2719 (1990). 16 Ha, T. et al. Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. U S A 93, 6264-6268 (1996). 17 Evans, G. W., Hohlbein, J., Craggs, T., Aigrain, L. Kapanidis, A. N. Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms. Nucleic Acids Res. 43, 5998-6008 (2016). 18 Santoso, Y. et al. Conformational transitions in DNA polymerase I revealed by single-molecule FRET. Proc. Natl Acad. Sci. U S A 107, 715-720 (2010). 19 Kenworthy, A. K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289-296 (2001). 20 Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515-527 (2006). 21 Kapanidis, A. N. et al. Alternating-laser excitation of single molecules. Acc. Chem. Res. 38, 523-533 (2005). 22 Friedman, L. J. Gelles, J. Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms. Methods 86, 27-36 (2015). 23 Larson, J. et al. Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat. Protoc. 9, 2317-2328 (2014). 24 Tetone, L. E. et al. Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue. Proc. Natl Acad. Sci. U S A 114, E1081-E1090 (2017). 25 Hohlbein, J., Craggs, T. D. Cordes, T. Alternating-laser excitation: single-molecule FRET and beyond. Chem. Soc. Rev. 43, 1156-1171 (2014). 26 Reyes-Lamothe, R., Sherratt, D. J. Leake, M. C. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498-501 (2010). 27 Mangiameli, S. M., Merrikh, C. N., Wiggins, P. A. Merrikh, H. Transcription leads to pervasive replisome instability in bacteria. Elife 6, e19848 (2017). 28 Ulbrich, M. H., Isacoff, E. Y. Subunit counting in membrane-bound proteins. Nat. Methods 4, 319-321 (2007). 29 Ogawa, T., Yu, X., Shinohara, A. Egelman, E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896-1899 (1993). 30 van der Heijden, T. et al. Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules. Nucleic Acids Res. 35, 5646-5657 (2007). 31 Modesti, M. et al. Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule. Structure 15, 599-609 (2007) 32 Liu, Y. et al. Conformational changes modulate the activity of human RAD51 protein. J Mol Biol.337, 817-827 (2004) 33 Roy, R., Hohng, S. Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507-516 (2008). 34 Lee, J. et al. Single-molecule Four-color FRET. Angew. Chem. Int. Ed. Engl. 49, 9922-9925 (2010) 35 Lin, Y.-H. Investigating How Mouse RAD51 Filament Dynamics Regulated by SWI5-SFR1 Complex Using Optical Tweezers Master thesis, National Taiwan University, (2016) | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70952 | - |
| dc.description.abstract | 真核細胞中，重組酶RAD51在DNA雙股斷裂的同源修復中扮演關鍵的角色，其調控步驟為RAD51核蛋白絲的穩定性。核蛋白絲的穩定性主要受到兩個機制調控，一則重組酶的核蛋白絲形成，另一則為重組酶自核蛋白絲上的解離。目前已知RAD51核蛋白絲的形成牽涉兩個步驟，即較緩慢的成核反應與蛋白絲延長反應，然而對重組酶在核蛋白絲的解離步驟卻瞭解甚少。本研究透過建立單分子螢光顯像平台，量測標記螢光分子的重組酶老鼠RAD51在ATP與AMPPNP兩種不同條件下核蛋白絲解離情形。實驗觀察到在ATP條件下，其核蛋白絲在觀測時間內完全解離比例極高，受限於其解離速率目前尚無法精準量測完整的解離過程；然而在水解被抑制的AMPPNP條件下，其核蛋白絲穩定性雖有提高，但不如預期的穩定，重組酶仍有一階或多階螢光變化。根據實驗的統計結果，螢光變化比例大致與螢光強度正相關，此一量測平台提供更清晰的重組酶動態變化，未來亦極具潛力研究輔助蛋白調控機制。 | zh_TW |
| dc.description.abstract | RAD51 plays a crucial role in homologous recombination to repair double strand break damage in eukaryotes. The stability of RAD51 protein filament is one of the most key regulatory processes. The formation of RAD51 protein filament includes two processes, slow nucleation and fast extension process. However, there is limited understanding of protein filament stability and protein dissociation kinetics.Here, we developed single-molecule fluorescence imaging platform to investigate the stability of labeled mouse RAD51 protein filament. We characterized the stability of RAD51 filament and RAD51 dissociation from the protein filament under two different conditions, ATP or AMPPNP. Most RAD51 filament under ATP fully dissociates within our observation time, which made us difficult to track the complete dissociation process, and thus we move to AMPPNP condition. Surprisingly, RAD51 filaments under AMPPNP condition, although expected stably bound, show one or multiple intensity drops, and the drop ratio is approximately positive correlated with the fluorescence intensity. This single-molecule fluorescence platform provides more detailed RAD51 filament dynamics, and has potential to be used to measure the regulation of accessory proteins. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:45:29Z (GMT). No. of bitstreams: 1 ntu-107-R05223187-1.pdf: 5859190 bytes, checksum: 9b201adb835b0fbd1bd5572ad0de8685 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 謝誌 i 摘要 iii Abstract iv 目錄 v 圖目錄 vii 表目錄 viii 第一章 緒論 1 1-1 雙股斷裂與同源重組修復 1 1-2 同源重組修復反應機制 1 1-2.1 RAD51核蛋白絲形成步驟 3 1-3 單分子螢光平台 6 1-4 研究動機 8 第二章 單分子多色螢光系統架設 9 2-1 單分子多色交替螢光基礎原理 9 2-2 多色雷射交替光路架設 9 2-2.1 顯微鏡激發光源標準調校方式 10 2-2.2 雷射機械快門速率分析 10 2-2.3 克卜勒式擴束器 11 2-2.4 可程式化激發切換 12 2-2.5 全內反射式物鏡系統 13 2-2.6 影像分光儀 13 2-2.7 電子倍增耦合偵測器 13 2-2.8 影像分析系統 13 第三章 實驗方法與設計 18 3-1 重組酶老鼠RAD51-Cy3蛋白的純化、標記與保存 18 3-2 重組酶老鼠RAD51螢光分子標定 18 3-3 DNA基質製備 19 3-3.1 單分子基質製備 19 3-3.2 單分子基質黏合反應配方 19 3-3.3 單分子基質黏合反應流程 19 3-4 緩衝溶液及反應溶液配方 20 3-5 除氧系統配置 20 3-6 反應玻片製備 21 3-6.1 玻片清洗 21 3-6.2 聚乙二醇表面修飾 22 3-6.3反應通道製作 23 3-7單分子多色螢光切換實驗流程 23 3-8 實驗設計 24 3-8.1 蛋白共位比例分析 24 3-8.2 單一重組酶解離動力學 24 3-8.3 重組酶核蛋白絲蛋白數量變化分析 25 第四章 實驗結果 26 4-1 影像對應與校正 26 4-1.1 單一分子螢光強度分析 26 4-1.2 非專一性蛋白吸附的分析 27 4-2 重組酶核蛋白絲螢光共位變化分析 29 4-3 重組酶核蛋白絲解離動態分析 30 4-4 AMPPNP重組酶核蛋白絲解離比率與螢光強度關係 36 4-5 以螢光強度變化值作為重組酶核蛋白絲分析的討論 41 4-5.1 蛋白螢光強度標準數值標定 41 4-5.2 重組酶核蛋白絲穩定性分析控制組 42 4-5.3 重組酶解離過程細節動態 43 第五章 結論與未來展望 44 5-1結論 44 5-2未來展望: 46 參考文獻 51 附錄 54 附錄一 實驗用藥品清單 54 附錄二 單分子光路系統濾鏡列表 55 | |
| dc.language.iso | zh-TW | |
| dc.subject | 單分子螢光顯微 | zh_TW |
| dc.subject | 同源重組修復 | zh_TW |
| dc.subject | RAD51 | zh_TW |
| dc.subject | homologous recombination repair | en |
| dc.subject | RAD51 | en |
| dc.subject | single-molecule fluorescence imaging | en |
| dc.title | 發展單分子螢光顯微平台研究RAD51重組酶核蛋白絲的解離動態 | zh_TW |
| dc.title | Developing Single-Molecule Fluorescence Imaging Platform to Study Dissociation Dynamics of RAD51 Recombinase Filament | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李以仁,范秀芳 | |
| dc.subject.keyword | 同源重組修復,RAD51,單分子螢光顯微, | zh_TW |
| dc.subject.keyword | homologous recombination repair,RAD51,single-molecule fluorescence imaging, | en |
| dc.relation.page | 55 | |
| dc.identifier.doi | 10.6342/NTU201802384 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-02 | |
| 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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