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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Hung-Yi Wu | en |
| dc.contributor.author | 吳泓儀 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:40:30Z | - |
| dc.date.available | 2017-09-02 | |
| dc.date.copyright | 2015-09-02 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-22 | |
| dc.identifier.citation | Cox, M. M. Motoring along with the bacterial RecA protein. Nat. Rev. Mol. Cell Biol. 8, 127–138 (2007).
2. Van Loenhout, M. T. J., van der Heijden, T., Kanaar, R., Wyman, C. & Dekker, C. Dynamics of RecA filaments on single-stranded DNA. Nucleic Acids Res. 37, 4089–4099 (2009). 3. Pugh, B. F. & Cox, M. M. General mechanism for RecA protein binding to duplex DNA. J. Mol. Biol. 203, 479–493 (1988). 4. 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). 5. Lusetti, S. L. et al. C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects. J. Biol. Chem. 278, 16372–16380 (2003). 6. Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006). 7. Kim, S. H. et al. Cooperative Conformational Transitions Keep RecA Filament Active During ATPase Cycle. J. Am. Chem. Soc. 136, 14796–14800 (2014). 8. Qi, Z. et al. DNA Sequence Alignment by Microhomology Sampling during Homologous Recombination. Cell 160, 856–869 (2015). 9. Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M. & Keck, J. L. SSB as an organizer/mobilizer of genome maintenance complexes. Crit. Rev. Biochem. Mol. Biol. 43, 289–318 (2008). 10. Lohman, T. M. & Ferrari, M. E. ESCHERICHIA COLI SINGLE? STRANDED DNA-BINDING PROTEIN: Multiple DNA-Binding Modes and Cooperativities. Annu. Rev. Biochem. 63, 527–570 (1994). 11. Zhou, R. et al. SSB functions as a sliding platform that migrates on DNA via reptation. Cell 146, 222–232 (2011). 12. Roy, R., Kozlov, A. G., Lohman, T. M. & Ha, T. SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461, 1092–1097 (2009). 13. Lee, K. S. et al. Ultrafast redistribution of E. coli SSB along Long single-stranded DNA via intersegment transfer. J. Mol. Biol. 426, 2413–2421 (2014). 14. Roy, R., Kozlov, A. G., Lohman, T. M. & Ha, T. Dynamic Structural Rearrangements Between DNA Binding Modes of E. coli SSB Protein. J. Mol. Biol. 369, 1244–1257 (2007). 15. Bhattacharyya, B. et al. Structural mechanisms of PriA-mediated DNA replication restart. Proc. Natl. Acad. Sci. U. S. A. 111, 1373–1378 (2014). 16. Morrical, S. W., Lee, J. & Cox, M. M. Continuous association of Escherichia coli single-stranded DNA binding protein with stable complexes of recA protein and single-stranded DNA. Biochemistry 25, 1482–1494 (1986). 17. Kowalczykowski, S. C. & Krupp, R. A. Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. J. Mol. Biol. 193, 97–113 (1987). 18. Bell, J. C., Plank, J. L., Dombrowski, C. C. & Kowalczykowski, S. C. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491, 274–278 (2012). 19. 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). 20. Hsu, H.-F., Ngo, K. V, Chitteni-pattu, S., Cox, M. M. & Li, H.-W. Investigating Deinococcus radiodurans RecA protein filament formation on dsDNA by a real-time single- molecule approach. Biochemistry 50, 8270–8280 (2011). 21. Tseng, T.-L. Investigating RecA Filament Assembly in the Presence of SSB at Single-Molecule Level. (National Taiwan University, 2013). 22. Kim, J.-Y., Kim, C. & Lee, N. K. Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering. Nat. Commun. 6, 6992 (2015). 23. Kowalczykowski, S. C., Clow, J., Somani, R. & Varghese, a. Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction. J. Mol. Biol. 193, 81–95 (1987). 24. Lavery, P. E. & Kowalczykowski, S. C. Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents. J. Biol. Chem. 267, 9307–9314 (1992). 25. Wu, H.-Y. & Li, H.-W. Crowding alters the dynamics and the length of RecA nucleoprotein filaments in RecA-mediated strand exchange. ChemPhysChem 15, 80–84 (2014). 26. Lesterlin, C., Ball, G., Schermelleh, L. & Sherratt, D. J. RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506, 249–53 (2014). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54112 | - |
| dc.description.abstract | RecA同源重組酶在DNA同源重組修復機制中負責催化同源配對和股交換反應。DNA修復的第一步，RecA必須和單股DNA結合蛋白 (SSB) 競爭single-stranded DNA (ssDNA) 以形成RecA核蛋白絲。過去的研究認為，RecA蛋白在這個過程中會採取較為被動的策略：等待直到一段ssDNA從SSB上裸露出來，接著在這段ssDNA上成核，然後延伸形成完整的核蛋白絲。在這個研究中，我們使用單分子拴球實驗 (TPM) 實時觀察單一RecA核蛋白絲在SSB-ssDNA上形成的過程。RecA核蛋白絲形成時會將ssDNA拉直，反應在拴球布朗運動的增加上。藉由改變DNA中ssDNA的長度 (60、65、70、72、75、78、80、90、100 nt) ，同時控制SSB的數量，使ssDNA上只會有一個65-nt mode SSB，我們發現RecA E38K的成核時間並沒有與ssDNA長度呈現顯著的負相關性。相反地，其成核時間隨著ssDNA上的SSB數目不同而改變 (70、135、200、264 nt，對應到1、2、3、4個SSB) 。當在反應中加入free SSB時，我們發現RecA E38K需要更長的成核時間和延伸時間，反應出來的拴球布朗運動改變量也比較小，意味著SSB構型轉換可能參與在RecA E38K的成核過程，同時影響成核速度、延伸速度與核蛋白絲長度。我們的實驗結果揭示了一個意想不到的RecA核蛋白絲形成機制：主動成核，其中包含了直接的RecA-SSB-ssDNA交互作用和SSB構型轉換。 | zh_TW |
| dc.description.abstract | RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments as the first step of the repair process. Previously, it has been suggested that RecA competes with SSB by binding and extending onto the free ssDNA region. In this study, we monitored individual RecA filament formation on SSB-ssDNA by tethered particle motion (TPM) technique in real-time. Binding of RecA on the SSB wrapped-ssDNA extends the DNA substrate, visible by the increase in bead Brownian motion in TPM imaging. By varying the length of ssDNA gap from 60 to 100 nucleotides (60, 65, 70, 72, 75, 78, 80, 90, 100), in which only one SSB binding is allowed using the 65mer wrapping mode, we found the nucleation time of RecA E38K to form extended nucleoprotein filament lacks apparent ssDNA gap size dependence. Instead, the nucleation time changes when DNA substrates with 1, 2, 3 and 4 SSB bound (70, 135, 200, 264 nucleotides) were used. Longer nucleation time and shorter RecA filament length were found when free SSB was present, indicating the involvement of SSB binding mode conversion. Our data have revealed an unexpected RecA filament formation mechanism on SSB-ssDNA in which direct RecA-SSB-ssDNA interaction and SSB binding mode conversion are involved. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:40:30Z (GMT). No. of bitstreams: 1 ntu-104-R02223101-1.pdf: 7069842 bytes, checksum: 3a0794a12df3224274fbec1ace3f0884 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 章節目錄
第一章 前言 5 1.1 RecA核蛋白絲：結構、動力學和功能 5 1.2 Single-stranded DNA binding protein (SSB) ：結構、動力學和功能 8 1.3 RecA核蛋白絲在SSB-ssDNA上之形成機制：已知與未知 12 1.4 本研究探討RecA核蛋白絲在SSB-ssDNA上之形成機制的切入點 17 第二章 實驗方法 19 2.1 製備微流體通道 19 2.2 製備streptavidin-coated聚苯乙烯小球 19 2.3 DNA合成 20 2.3.1 dsDNA 21 2.3.2 T-gap DNA 21 2.3.3 AC-gap DNA 24 2.4 製備Anti-digoxigenin溶液 27 2.5 製備RecA反應緩衝溶液 27 2.6 單分子拴球實驗步驟 28 2.7 數據分析和Matlab程式碼 29 2.7.1 Time course實驗 29 2.7.2 Histogram實驗 31 第三章 數據結果與討論 32 3.1 SSB-ssDNA大幅抑制RecA E38K核蛋白絲形成 32 3.2 改變單一SSB旁邊的ssDNA長度並不會顯著改變RecA E38K成核時間 35 3.3 RecA E38K成核時間和ssDNA上SSB的數目有關 37 3.4 溶液中的SSB會大幅增加RecA E38K成核時間 42 3.5 RecA在SSB-ssDNA上形成核蛋白絲的主動成核機制 44 第四章 結論與未來展望 46 參考文獻 50 附錄 53 試劑與耗材 53 DNA引子序列 55 | |
| dc.language.iso | zh-TW | |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | 延伸 | zh_TW |
| dc.subject | 成核 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | 延伸 | zh_TW |
| dc.subject | 成核 | zh_TW |
| dc.subject | Homologous recombination | en |
| dc.subject | SSB | en |
| dc.subject | extension | en |
| dc.subject | RecA | en |
| dc.subject | extension | en |
| dc.subject | filament | en |
| dc.subject | filament | en |
| dc.subject | RecA | en |
| dc.subject | SSB | en |
| dc.subject | Homologous recombination | en |
| dc.subject | nucleation | en |
| dc.subject | nucleation | en |
| dc.title | RecA核蛋白絲於SSB-ssDNA上的形成過程包含RecA-SSB交互作用與SSB構型轉換 | zh_TW |
| dc.title | RecA Filament Assembly on SSB-wrapped ssDNA Includes RecA-SSB interactions and SSB Binding Mode Conversion | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 謝道時(Tao-Shih Hsieh),冀宏源(Hung-Yuan (Peter),李以仁(I-Ren Lee) | |
| dc.subject.keyword | 同源重組,成核,延伸, | zh_TW |
| dc.subject.keyword | RecA,SSB,Homologous recombination,nucleation,extension,filament, | en |
| dc.relation.page | 55 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-22 | |
| 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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