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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Hung-Chi Liu | en |
| dc.contributor.author | 劉泓其 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:45:21Z | - |
| dc.date.available | 2018-02-02 | |
| dc.date.copyright | 2016-02-02 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-12-01 | |
| dc.identifier.citation | 1 Yin, H., Landick, R. & Gelles, J. Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. Biophys. J. 67, 2468-2478 (1994).
2 Modrich, P. & Lahue, R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101-133 (1996). 3 Kowalczykowski, S. C. Initiation of genetic recombination and recombination-dependent replication. Trends Biochem. Sci. 25, 156-165 (2000). 4 Jeong, C. et al. MutS switches between two fundamentally distinct clamps during mismatch repair. Nature Struct. Biol. 18, 379-385 (2011). 5 Lamers, M. H. et al. The crystal structure of DNA mismatch repair protein MutS binding to a G.T mismatch. Nature 407, 711-717 (2000). 6 Lusetti, S. L. & Cox, M. M. The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. Annu. Rev. Biochem. 71, 71-100 (2002). 7 Ngo, K. V., Molzberger, E. T., Chitteni-Pattu, S. & Cox, M. M. Regulation of Deinococcus radiodurans RecA protein function via modulation of active and inactive nucleoprotein filament states. J. Biol. Chem. 288, 21351-21366 (2013). 8 Fan, H.-F., Cox, M. M. & Li, H.-W. Developing single-molecule TPM experiments for direct observation of successful RecA-mediated strand exchange reaction. PLoS ONE 6, e21359 (2011). 9 Cox, M. M. Motoring along with the bacterial RecA protein. Nature Rev. Mol. Cell Biol. 8, 127-138 (2007). 10 Constantin, N., Dzantiev, L., Kadyrov, F. A. & Modrich, P. Human mismatch repair: reconstitution of a nick-directed bidirectional reaction. J. Biol. Chem. 280, 39752-39761 (2005). 11 Li, G.-M. Mechanisms and functions of DNA mismatch repair. Cell Res. 18, 85-98 (2008). 12 Grilley, M., Griffith, J. & Modrich, P. Bidirectional excision in methyl-directed mismatch repair. J. Biol. Chem. 268, 11830-11837 (1993). 13 Su, S. S. & Modrich, P. Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs. Proc. Natl. Acad. Sci. USA 83, 5057-5061 (1986). 14 Matson, S. W. & Robertson, A. B. The UvrD helicase and its modulation by the mismatch repair protein MutL. Nucleic Acids Res. 34, 4089-4097 (2006). 15 Rayssiguier, C., Thaler, D. S. & Radman, M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342, 396-401 (1989). 16 Worth, L., Clark, S., Radman, M. & Modrich, P. Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs. Proc. Natl. Acad. Sci. USA 91, 3238-3241 (1994). 17 Tham, K.-C. et al. Mismatch repair inhibits homeologous recombination by coordinated directional unwinding of trapped DNA structures. Mol. Cell 51, 326-337 (2013). 18 Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nature Methods 5, 507-516 (2008). 19 Ritchie, D. B. & Woodside, M. T. Probing the structural dynamics of proteins and nucleic acids with optical tweezers. Curr. Opin. Struct. Biol. 34, 43-51 (2015). 20 Yan, J., Skoko, D. & Marko, J. F. Near-field-magnetic-tweezer manipulation of single DNA molecules. Phys.l Rev. E 70, 011905 (2004). 21 Schafer, D. A., Gelles, J., Sheetz, M. P. & Landick, R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 352, 444-448 (1991). 22 Winkler, I. et al. Chemical trapping of the dynamic MutS-MutL complex formed in DNA mismatch repair in Escherichia coli. J. Biol. Chem. 286, 17326-17337 (2011). 23 Cho, W.-K. et al. ATP alters the diffusion mechanics of MutS on mismatched DNA. Structure 20, 1264-1274. 24 Bjornson, K. P., Allen, D. J. & Modrich, P. Modulation of MutS ATP hydrolysis by DNA Cofactors. Biochemistry 39, 3176-3183 (2000). 25 Harris, D. C. Quantitative chemical analysis, 7th ed., W.H. Freeman, New York. (2007). 26 Sagi, D., Tlusty, T. & Stavans, J. High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity. Nucleic Acids Res. 34, 5021-5031 (2006). 27 Markowski, C. A. & Markowski, E. P. Conditions for the Effectiveness of a Preliminary Test of Variance. Am. Stat. 44, 322-326 (1990). 28 Bazemore, L. R., Folta-Stogniew, E., Takahashi, M. & Radding, C. M. RecA tests homology at both pairing and strand exchange. Proc. Natl. Acad. Sci. USA 94, 11863-11868 (1997). 29 Acharya, S., Foster, P. L., Brooks, P. & Fishel, R. The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair. Mol. Cell 12, 233-246 (2003). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51702 | - |
| dc.description.abstract | 在DNA複製的過程中，可能會受到一些破壞而需要修復機制，而同源重組反應即是其中一種重要的修復機制。在大腸桿菌等原核生物中，其中核心的步驟在於RecA蛋白與DNA形成的核絲結構可辨認相同序列的DNA分子，並透過股交換反應來修復DNA。先前的研究指出，即使在欲交換的DNA序列上面有與原本單股DNA有不配對的序列，仍可進行部分同源股交換反應，生成含有錯誤配對的DNA產物。而錯誤辨認蛋白MutS可以抑制這樣的部分同源股交換反應，但其中抑制的原理以及機制卻仍不明。除了傳統的電泳生化分析技術，我們也使用了單分子栓球實驗去觀察部分同源股交換反應，以及MutS蛋白在其中的角色。首先，我們發現在進行RecA催化部分同源股交換反應當中，即使在接近的錯誤配對比例之下，錯誤配對的連續性是影響RecA催化部分同源股交換反應的重要因素。第二，我們使用線性且較短的DNA基質組進行MutS蛋白抑制部分同源股交換反應，除了反應被抑制以外，也看見更高分子量的副產物出現，我們推測此副產物可能是股交換反應的中間產物，三股DNA的結構。接著，我們也以單分子栓球實驗去測量RecA核蛋白絲與雙股DNA的作用，結果顯示在我們的解析度之下，MutS蛋白在這一步驟並無影響，這一系列的實驗也提示了我們三股DNA是MutS蛋白抑制部分同源股交換反應的研究方向。 | zh_TW |
| dc.description.abstract | Homologous recombination (HR) plays an important role in meiosis and DNA damage repair. In prokaryotes, RecA nucleoprotein filaments search complementary DNA sequence, and carry out the strand-exchange reaction of essential steps in HR. Moreover, RecA can catalyze strand-exchange reaction even if DNA sequence not totally homologous, i.e. homeologous. Previous studies indicate that mismatch repair protein, MutS, inhibits RecA-mediated homeologous recombination. However, the inhibition mechanism remains unknown. We found the DNA mismatch density plays an important role in inhibiting the homeologous strand-exchange reaction. Using gel-shift electrophoresis assay, an intermediate state of strand exchange reaction with higher molecular weight appeared in the presence of MutS. This suggests that MutS proteins promote the accumulation of the reaction intermediate, thus reduce final reaction products. Using single-molecule tethered particle motion experiments, we measured interaction between RecA filaments and homeologous DNA substrates in the presence of MutS. These experiments offer information about how MutS inhibition might take place. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:45:21Z (GMT). No. of bitstreams: 1 ntu-104-R01223179-1.pdf: 2481568 bytes, checksum: a49ae7cde058715f687265cfe0a444dc (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目 錄........III
圖目錄.........V 表目錄.......VII 摘要........VIII Abstract......IX 第一章 緒論 1.1 同源重組與RecA重組酶.....................................1 1.2 DNA錯誤配對修復與MutS錯誤辨認蛋白........................3 1.3 MutS抑制RecA重組酶催化部分同源重組反應...................6 1.4 單分子栓球實驗...........................................7 第二章 實驗架設與方法 2.1 MutS蛋白純化.............................................8 2.1.1 氯化銫梯度(CsCl banding)離心法純化質體................10 2.1.2 大腸桿菌培養以及MutS表達測試..........................13 2.1.3 鹽析與親和性管柱純化..................................18 2.1.4 MutS活性測試與濃度測量................................24 2.2 fd 噬菌體質體DNA純化....................................28 2.3 DNA合成.................................................30 2.3.1以基因合成設計部分同源DNA..............................30 2.3.2以fd噬菌體與M13mp18噬菌體質體DNA設計部分同源DNA........34 2.4 RecA重組酶催化股交換反應................................37 2.5單分子栓球實驗...........................................39 2.6 數據處理................................................42 第三章 結果與討論 3.1 部分同源股交換反應......................................43 3.1.1基因合成DNA進行部分同源股交換反應......................43 3.1.2 fd噬菌體與M13mp18噬菌體質體DNA進行部分同源股交換反應..44 3.2 單分子栓球實驗..........................................48 3.2.1 以離去比例計算反應效率................................48 3.2.2 MutS蛋白影響RecA蛋白絲與雙股DNA的結合.................51 第四章 結論與未來展望 4.1 RecA催化部分同源股交換的反應效率........................58 4.2 MutS如何抑制RecA催化部分同源股交換反應..................60 4.3 未來展望................................................62 參考文獻....................................................63 圖目錄 圖1-1 同源重組中間的步驟以及參與的蛋白質 2 圖1-2 大腸桿菌內的MMR模型示意圖 4 圖1-3 大腸桿菌MutS蛋白結構圖 5 圖1-4 MutS蛋白結合ATP形成滑動夾子 5 圖1-5 布朗運動反映不同雙股DNA的長度 7 圖2-1 小量蛋白質溶液鹽析測試 20 圖2-3 MutS純化後的SDS-PAGE圖 22 圖2-2以肝素親合管柱純化MutS溶液 22 圖2-4 以肝素親合管柱純壞MutS的UV-Vis圖 23 圖2-5 MutS ATP水解酶活性實驗原始數據 27 圖2-6 MutS ATP水解酶活性實驗處理過數據 27 圖2-7 以不同限制酶鑑定噬菌體fd 的RF質體DNA 29 圖2-8 3χF3χH與pUC57-kan-mis300質體DNA的錯誤配對設計 31 圖2-9 ds150與ss231-mis進行股交換反應圖示 32 圖2-10 以fd、M13mp18設計的289-209 DNA基質組 34 圖2-11 以fd、M13mp18設計的422-340 DNA基質組 35 圖2-12 RecA重組酶催化股交換反應 37 圖2-13單分子栓球實驗架設 40 圖3-1 231/150 基因合成DNA進行RecA催化股交換反應 43 圖3-2 289/209 噬菌體DNA進行RecA催化股交換反應 45 圖3-3 422/340噬菌體DNA進行RecA催化股交換反應 45 圖3-4 MutS蛋白抑制部分同源股交換反應 47 圖3-5 單分子栓球離去實驗效率柱狀圖 50 圖3-6 Control未離開栓球的時間軌跡 52 圖3-7 Homo未離開栓球的時間軌跡 53 圖3-8 HomoM未離開栓球的時間軌跡 54 圖3-9 Homeo未離開栓球的時間軌跡 55 圖3-10 HomeoM未離開栓球的時間軌跡 56 圖4-1 MutS蛋白於單分子栓球實驗的3股DNA假設 61 圖4-2 單分子螢光栓球實驗與MutS蛋白 62 表目錄 表2-2 純化MutS 所需要的所有緩衝液 9 表2-1 純化MutS蛋白的流程 9 表2-3 LB培養基平板的製備配方 13 表2-4 5x SDS sample buffer的製備配方 17 表2-5 CBB染劑的製備配方 17 表2-6 退染溶液的製備配方 17 表2-7 AT水解酶活性偵測實驗條件 26 表2-8 ds150與ss231-mis的製備條件 33 表2-9 fd, M13mp18 DNA基質的製備條件 36 表2-10 股交換反應與終止反應條件 38 表2-11 混合DNA (hybrid DNA) 的製備條件 41 表3-1 單分子栓球離去實驗效率與誤差 49 表3-2 學生t檢定結果 50 表3-3 未離開栓球的時間軌跡分析 57 | |
| dc.language.iso | zh-TW | |
| dc.subject | DNA 修復 | zh_TW |
| dc.subject | 部分同源重組反應 | zh_TW |
| dc.subject | 單分子栓球實驗 | zh_TW |
| dc.subject | RecA 重組? | zh_TW |
| dc.subject | 單分子 | zh_TW |
| dc.subject | MutS蛋白 | zh_TW |
| dc.subject | Homeologous recombination | en |
| dc.subject | TPM | en |
| dc.subject | Single molecule | en |
| dc.subject | DNA repair | en |
| dc.subject | MutS | en |
| dc.subject | RecA | en |
| dc.title | 單分子技術觀察DNA錯誤配對辨認蛋白 MutS 如何抑制 RecA 重組酶進行部分同源股交換反應 | zh_TW |
| dc.title | Investigate How Mismatch Repair Protein MutS Inhibits RecA-Catalyzed Homeologous Strand-Exchange Reaction Using a Single-Molecule Method | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張煥宗(Huan-Tsung Chang),冀宏源(Hung-Yuan Chi) | |
| dc.subject.keyword | RecA 重組?,單分子栓球實驗,單分子,DNA 修復,MutS蛋白,部分同源重組反應, | zh_TW |
| dc.subject.keyword | RecA,TPM,Single molecule,DNA repair,MutS,Homeologous recombination, | en |
| dc.relation.page | 64 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-12-01 | |
| 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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