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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Ting-Tzu Chang | en |
| dc.contributor.author | 張庭慈 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:18:37Z | - |
| dc.date.available | 2021-08-25 | |
| dc.date.copyright | 2016-08-25 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-18 | |
| dc.identifier.citation | 1 Story, R. M., Weber, I. T. & Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318-325 (1992).
2 Story, R. M. & Steitz, T. A. Structure of the recA protein-ADP complex. Nature 355, 374-376 (1992). 3 Cox, M. M. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol 8, 127-138 (2007). 4 Di Capua, E., Engel, A., Stasiak, A. & Koller, T. Characterization of complexes between recA protein and duplex DNA by electron microscopy. J Mol Biol 157, 87-103 (1982). 5 Egelman, E. H. & Yu, X. The location of DNA in RecA-DNA helical filaments. Science 245, 404-407 (1989). 6 Little, J. W. Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie 73, 411-421 (1991). 7 Goodman, M. F. Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu Rev Biochem 71, 17-50 (2002). 8 Wyman, C., Ristic, D. & Kanaar, R. Homologous recombination-mediated double-strand break repair. DNA Repair (Amst) 3, 827-833 (2004). 9 Galletto, R., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875-878 (2006). 10 Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515-527 (2006). 11 Cox, J. M., Tsodikov, O. V. & Cox, M. M. Organized unidirectional waves of ATP hydrolysis within a RecA filament. PLoS Biol 3, e52 (2005). 12 Pugh, B. F. & Cox, M. M. Stable binding of recA protein to duplex DNA. Unraveling a paradox. J Biol Chem 262, 1326-1336 (1987). 13 Pugh, B. F. & Cox, M. M. General mechanism for RecA protein binding to duplex DNA. J Mol Biol 203, 479-493 (1988). 14 Roca, A. I. & Singleton, S. F. Direct evaluation of a mechanism for activation of the RecA nucleoprotein filament. J Am Chem Soc 125, 15366-15375 (2003). 15 Arenson, T. A., Tsodikov, O. V. & Cox, M. M. Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA. J Mol Biol 288, 391-401 (1999). 16 Kim, J. I., Cox, M. M. & Inman, R. B. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four-strand exchanges. J Biol Chem 267, 16444-16449 (1992). 17 Shan, Q. & Cox, M. M. On the mechanism of RecA-mediated repair of double-strand breaks: no role for four-strand DNA pairing intermediates. Mol Cell 1, 309-317 (1998). 18 Chow, S. A., Chiu, S. K. & Wong, B. C. RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA. J Mol Biol 223, 79-93 (1992). 19 Chen, Z., Yang, H. & Pavletich, N. P. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489-484 (2008). 20 Ennis, D. G., Levine, A. S., Koch, W. H. & Woodgate, R. Analysis of recA mutants with altered SOS functions. Mutat Res 336, 39-48 (1995). 21 Witkin, E. M., McCall, J. O., Volkert, M. R. & Wermundsen, I. E. Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol Gen Genet 185, 43-50 (1982). 22 Cazaux, C., Mazard, A. M. & Defais, M. Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele. Mol Gen Genet 240, 296-301 (1993). 23 Bar-Ziv, R. & Libchaber, A. Effects of DNA sequence and structure on binding of RecA to single-stranded DNA. Proc Natl Acad Sci U S A 98, 9068-9073 (2001). 24 Lavery, P. E. & Kowalczykowski, S. C. Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins. J Biol Chem 267, 20648-20658 (1992). 25 Morrical, S. W. & Cox, M. M. Stabilization of recA protein-ssDNA complexes by the single-stranded DNA binding protein of Escherichia coli. Biochemistry 29, 837-843 (1990). 26 Lavery, P. E. & Kowalczykowski, S. C. A postsynaptic role for single-stranded DNA-binding protein in recA protein-promoted DNA strand exchange. J Biol Chem 267, 9315-9320 (1992). 27 Britt, R. L. et al. Disassembly of Escherichia coli RecA E38K/DeltaC17 nucleoprotein filaments is required to complete DNA strand exchange. J Biol Chem 285, 3211-3226 (2010). 28 Beernink, H. T. & Morrical, S. W. RMPs: recombination/replication mediator proteins. Trends Biochem Sci 24, 385-389 (1999). 29 Eggler, A. L., Lusetti, S. L. & Cox, M. M. The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein. J Biol Chem 278, 16389-16396 (2003). 30 Tseng, T.-L. Investigating RecA filament assembly in the presence of SSB at single-molecule level Master thesis, National Taiwan University, (2013). 31 Lin, H.-C. UV-resistant RecA mutant E38K has small ssDNA binding unit: single molecule FRET studies Master thesis, National Taiwan University, (2014). 32 Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94, 1826-1835 (2008). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49185 | - |
| dc.description.abstract | RecA蛋白在雙股DNA斷裂所引發的同源重組修復中扮演相當關鍵的角色，在同源重組過程中，RecA核蛋白絲的形成被認為是同源重組過程中的重要調控步驟，而形成RecA核蛋白絲主要包含緩慢的成核作用以及快速的延伸作用。核蛋白絲會在其他輔助蛋白的協助下，尋找未受損的雙股DNA之互補序列，並進行股交換反應以修復受損的DNA。研究發現在高劑量紫外光照射下存活率較高的RecA蛋白突變型E38K，比起RecA蛋白能在更短的時間內與單股DNA進行成核反應。在本論文中，利用單分子螢光共振能量轉移的實驗以及隱馬可夫模型，研究RecA蛋白和RecA蛋白突變型E38K在不同長度的單股DNA上之成核作用，而實驗結果發現比起RecA蛋白，RecA蛋白突變型E38K在相同長度的單股DNA上有較高的成核頻率，且可以透過更小的成核單位與單股DNA結合。此外，實驗結果也顯示RecA蛋白突變型E38K可能以單體為單位，從穩定的核蛋白絲尾端脫離或結合。 | zh_TW |
| dc.description.abstract | RecA plays a key role in homologous recombination pathway to repair double-stranded DNA break damage. In homologous recombination, the assembly of RecA onto the ssDNA to form nucleoprotein filaments includes a slow nucleation and a rapid extension step. The assembly of RecA-ssDNA nucleoprotein filaments is the key regulatory step for homologous recombination, and the assembled nucleoprotein filament is responsible for mediating DNA homology search and downstream strand exchange. The previous study showed that a recA strain surviving high doses of UV radiation includes a dominative E38K point mutation. Single-molecule biochemical studies showed that E38K mutants have faster kinetics in forming nucleoprotein filaments compared to wild-type RecA. In this study, we used a single-molecule fluorescence resonance energy transfer (smFRET) experiment and hidden Markov analysis to study the assembly of wild-type RecA and RecA E38K at different ssDNA length (for wild-type RecA, (dT)n, n = 19, 21, 23, 27; for RecA E38K, (dT)n, n = 9, 16-23). We found that RecA E38K has smaller nucleation unit and higher nucleation frequency at the same ssDNA length ((dT)n, n = 19, 21, 23) compared to wild-type RecA. We also showed that RecA E38K filament likely assemble and disassemble with one monomer at the 5′ end of nucleoprotein filament. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:18:37Z (GMT). No. of bitstreams: 1 ntu-105-R03223120-1.pdf: 3688183 bytes, checksum: d6bb7f5176e46b6055bcdee4b3cc6264 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 摘要 ii
Abstract iii 圖目錄 vii 表目錄 ix 第1章 緒論 1 1-1 RecA蛋白 1 1-1.1 RecA的結構 1 1-1.2 RecA的功能 2 1-2 RecA蛋白突變型E38K 5 1-2.1 RecA E38K之結構 5 1-2.2 RecA E38K之特性 6 1-3 研究動機 10 第2章 實驗方法與設計 12 2-1 蛋白質的來源 12 2-2 DNA基質設計 12 2-3 溶液配方及製備 14 2-3.1 緩衝溶液及反應溶液配方 14 2-3.2 除氧系統 (oxygen scavenging system) 配置 14 2-3.3 蛋白質的反應條件 15 2-4 反應玻片製備 15 2-4.1 玻片清洗 15 2-4.2 聚乙二醇修飾 16 2-4.3 反應通道製作 18 2-5 單分子螢光實驗之架設 18 2-5.1 光學顯微鏡架設 18 2-5.2 EMCCD參數設定 20 2-5.3 系統測試 20 2-6 單分子螢光實驗流程 24 2-7 數據分析 25 2-7.1 影像的Cy3-Cy5分子配對 25 2-7.2 單分子時間軌跡 26 2-7.3 隱馬可夫分析 27 2-7.4 成核次數與頻率 27 2-7.5 停留時間 27 2-7.6 躍遷密度圖 27 第3章 實驗結果與討論 29 3-1 RecA與RecA E38K在ssDNA之成核反應 29 3-1.1 成核反應之時間軌跡 29 3-1.2 成核頻率 31 3-2 RecA E38K成核反應之速率常數及平衡常數 36 3-2.1 停留時間 36 3-2.2 速率常數 37 3-2.3 平衡常數 38 3-3 RecA E38K形成核蛋白絲時5′ 端之動力表現 40 3-3.1 時間軌跡 40 3-3.2 躍遷密度圖 42 第4章 實驗總結與未來展望 44 4-1 實驗總結 44 4-2 未來展望 46 參考文獻 48 附錄 51 附錄一、實驗使用之藥品清單 51 附錄二、RecA在ssDNA上進行成核反應之時間軌跡 53 附錄三、RecA E38K在ssDNA上進行成核反應之時間軌跡 57 附錄四、停留時間之數據整理 67 | |
| dc.language.iso | zh-TW | |
| dc.subject | RecA | zh_TW |
| dc.subject | 單分子螢光共振能量轉移 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | RecA蛋白突變型E38K | zh_TW |
| dc.subject | smFRET | en |
| dc.subject | RecA E38K (recA730) | en |
| dc.subject | homologous recombination | en |
| dc.subject | RecA | en |
| dc.title | 利用單分子螢光共振能量轉移研究RecA E38K核蛋白絲之動力學 | zh_TW |
| dc.title | Single-Molecule FRET Studies on Nucleoprotein Filament Dynamics of RecA E38K Mutant Protein | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王廷方,冀宏源,溫進德,李以仁 | |
| dc.subject.keyword | RecA,RecA蛋白突變型E38K,同源重組,單分子螢光共振能量轉移, | zh_TW |
| dc.subject.keyword | RecA,RecA E38K (recA730),homologous recombination,smFRET, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU201603371 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-20 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| Appears in Collections: | 化學系 | |
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| ntu-105-1.pdf Restricted Access | 3.6 MB | Adobe PDF |
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