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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Yi-Han Lu | en |
| dc.contributor.author | 呂易翰 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:01:48Z | - |
| dc.date.copyright | 2013-08-23 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-15 | |
| dc.identifier.citation | 1 Kleckner, N. Meiosis: how could it work? Proc. Natl. Acad. Sci. USA 93, 8167-8174 (1996).
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Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing. Genes Dev. 21, 1758-1766 (2007). 27 San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229-257 (2008). 28 Cloud, V., Chan, Y. L., Grubb, J., Budke, B. & Bishop, D. K. Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science 337, 1222-1225 (2012). 29 Habu, T., Taki, T., West, A., Nishimune, Y. & Morita, T. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucleic Acids Res. 24, 470-477 (1996). 30 Thorslund, T., Esashi, F. & West, S. C. Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1. EMBO J. 26, 2915-2922 (2007). 31 Okutsu, T. et al. Molecular cloning and characterization of Dmc1, a gene involved in gametogenesis, from the whiteleg shrimp Litopenaeus vannamei. Fisheries Science 76, 961-969 (2010). 32 Yoshida, K. et al. The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol. Cell1, 707-718 (1998). 33 Bannister, L. A. et al. A dominant, recombination-defective allele of Dmc1 causing male-specific sterility. PLoS Biol. 5, e105 (2007). 34 Chang, Y. C. et al. Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex. Biochemistry 44, 6052-6058 (2005). 35 Okorokov, A. L. et al. Structure of the hDmc1-ssDNA filament reveals the principles of its architecture. PLoS One 5, e8586 (2010). 36 Sheridan, S. D. et al. A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments. Nucleic Acids Res. 36, 4057-4066 (2008). 37 Yu, X. & Egelman, E. H. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale. J. Mol. Biol. 401, 544-551 (2010). 38 Masson, J. Y. et al. The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J. 18, 6552-6560 (1999). 39 Passy, S. I. et al. Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. USA 96, 10684-10688 (1999). 40 Bockelmann, U. Single-molecule manipulation of nucleic acids. Curr. Opin. Struct. Biol. 14, 368-373 (2004). 41 Gosse, C. & Croquette, V. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314-3329 (2002). 42 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). 43 Chung, C. & Li, H. W. Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases. J. Am. Chem. Soc. 135, 8920-8925 (2013). 44 Fan, H. F. & Li, H. W. Studying RecBCD helicase translocation along Chi-DNA using tethered particle motion with a stretching force. Biophys. J. 96, 1875-1883 (2009). 45 Chi, P. et al. Functional interactions of meiotic recombination factors Rdh54 and Dmc1. DNA Repair (Amst) 8, 279-284 (2009). 46 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). 47 Lee, M. H. et al. Calcium ion promotes yeast Dmc1 activity via formation of long and fine helical filaments with single-stranded DNA. J. Biol. Chem. 280, 40980-40984 (2005). 48 Bugreev, D. V., Golub, E. I., Stasiak, A. Z., Stasiak, A. & Mazin, A. V. Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J. Biol. Chem. 280, 26886-26895 (2005). 49 Hsu, H. F., Ngo, K. V., Chitteni-Pattu, S., Cox, M. M. & Li, H. W. Investigating Deinococcus radiodurans RecA protein filament formation on double-stranded DNA by a real-time single-molecule approach. Biochemistry 50, 8270-8280 (2011). 50 Vipond, I. B., Baldwin, G. S. & Halford, S. E. Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases. Biochemistry 34, 697-704 (1995). 51 Bailey, R. E., Smith, A. M. & Nie, S. Quantum dots in biology and medicine. Physica E 25, 1-12 (2004). 52 Li, Z., Golub, E. I., Gupta, R. & Radding, C. M. Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein. Proc. Natl. Acad. Sci. USA 94, 11221-11226 (1997). 53 Chi, P., San Filippo, J., Sehorn, M. G., Petukhova, G. V. & Sung, P. Bipartite stimulatory action of the Hop2-Mnd1 complex on the Rad51 recombinase. Genes Dev. 21, 1747-1757 (2007). 54 Pezza, R. J., Petukhova, G. V., Ghirlando, R. & Camerini-Otero, R. D. Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. J. Biol. Chem. 281, 18426-18434 (2006). 55 Leu, J. Y., Chua, P. R. & Roeder, G. S. The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes. Cell 94, 375-386 (1998). 56 Tsubouchi, H. & Roeder, G. S. The importance of genetic recombination for fidelity of chromosome pairing in meiosis. Dev. Cell 5, 915-925 (2003). 57 Petukhova, G. V., Romanienko, P. J. & Camerini-Otero, R. D. The Hop2 protein has a direct role in promoting interhomolog interactions during mouse meiosis. Dev. Cell 5, 927-936 (2003). 58 Rabitsch, K. P. et al. A screen for genes required for meiosis and spore formation based on whole-genome expression. Curr. Biol. 11, 1001-1009 (2001). 59 Gerton, J. L. & DeRisi, J. L. Mnd1p: an evolutionarily conserved protein required for meiotic recombination. Proc. Natl. Acad. Sci. USA 99, 6895-6900 (2002). 60 Tsubouchi, H. & Roeder, G. S. The Mnd1 protein forms a complex with hop2 to promote homologous chromosome pairing and meiotic double-strand break repair. Mol. Cell Biol.22, 3078-3088 (2002). 61 Saito, T. T., Tougan, T., Kasama, T., Okuzaki, D. & Nojima, H. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination. Nucleic Acids Res. 32, 3325-3339 (2004). 62 Chen, Y. K. et al. Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation. Proc. Natl. Acad. Sci. USA 101, 10572-10577 (2004). 63 Petukhova, G. V. et al. The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination. Nat. Struct. Mol. Biol. 12, 449-453 (2005). 64 Pezza, R. J., Camerini-Otero, R. D. & Bianco, P. R. Hop2-Mnd1 condenses DNA to stimulate the synapsis phase of DNA strand exchange. Biophys. J. 99, 3763-3772 (2010). 65 Bianco, P. R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374-378 (2001). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17224 | - |
| dc.description.abstract | 當 DNA 因為雙股斷裂 (double-strand break, DSB) 而受損時，細胞會進行一個稱為同源重組(homologous recombination, HR) 的反應來對受損的 DNA 進行修復。同源重組反應是由同源重組酶催化進行的。當此反應進行時，同源重組酶須要先和 ATP 結合，接著聚集在單股 DNA (single-stranded DNA, ssDNA) 上形成核蛋白絲結構。股交換反應完成後，同源重組酶會水解 ATP 接著從 DNA 上脫離。Dmc1在真核細胞內是專門負責進行減數分裂時期同源重組的蛋白質。和其他兩個會形成延長的核蛋白絲的同源蛋白質 RecA 及Rad51 不同的是，Dmc1 在利用電子顯微鏡的研究中被觀察到有兩種不同的結構，其一是延長的核蛋白絲 (extended nucleoprotein filament)，另一種則是以堆疊環狀 (stacked ring) 的型式和 DNA 結合。在本研究中，我們使用單分子拴球技術 (tethered particle motion, TPM) 來觀察Dmc1 在催化股交換反應進行時的即時狀態，並發現 Dmc1 於此過程中並不會造成延長的三股 DNA 中間產物結構產生。即使我們加入可以藉由抑制 ATP 水解而穩定核蛋白絲結構的鈣離子也不會影響此結果。 我們更進一步的利用自相關函數來分析反應時的時間對布朗運動趨勢圖來證實了 DNA 和 Dmc1 的交互作用。 | zh_TW |
| dc.description.abstract | Cells utilize homologous recombination (HR) to repair the DNA double-strand break (DSB). The reaction is conducted by a kinds of protein called recombinase. While conducting the HR reaction, recombinase need to bind with ATP and then assemble on single-stranded (ss) DNA to form nucleoprotein complex. After the strand exchange completed, ATP was hydrolyzed and recombinase disassemble from DNA. Dmc1 is a meiosis-specific recombinase responsible for HR in eukaryotic cell. Unlike other two Dmc1 homolog RecA and Rad51, which have been proved to form an extended nucleoprotein filament, Dmc1 was showed to have two kinds of structure of nucleoprotein filament by electron microscopy studies, one is extended nucleoprotein filament and another is stacked ring structure. In this study we use tethered particle motion (TPM) to observe the real time behavior while Dmc1-catalyzed strand exchange reaction was processing and found that Dmc1 does not form an extended three strand intermediate, even though we add more calcium to inhibit ATP hydeolysis and stabilize the Dmc1 filament. So we further utilize the autocorrelation function to analyze the reaction timetrace and prove the DNA-protein interaction. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:01:48Z (GMT). No. of bitstreams: 1 ntu-102-R00223210-1.pdf: 11450933 bytes, checksum: 75f4684585cc3f038331c9cb63a4bc5e (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目錄
第一章 緒論 1 1-1 減數分裂和染色體互換 1 1-2 同源重組 2 1-3 Dmc1蛋白 4 1-4 單分子拴球實驗 7 1-5 研究動機 8 第二章 實驗架構 9 2-1 蛋白質的表現及純化 9 2-2 DNA的製備 9 2-3 玻片之表面修飾 13 2-3-1 Flow chamber的製備 13 2-3-2 製備表面修飾streptavidin的220奈米聚苯乙烯球 13 2-3-3 玻片表面修飾及雙股DNA的固定 14 2-4 股交換離去反應 16 2-5 影像擷取及數據分析 17 第三章 實驗結果與討論 18 3-1 鈣離子濃度對Dmc1股交換活性的影響 18 3-1-1 實驗設計 18 3-1-2 鈣離子濃度不影響控制組實驗中拴球離去的比例 18 3-1-3 不同鈣離子濃度中Dmc1的股交換活性 20 3-2 Dmc1催化股交換反應時和DNA的結合情形 21 3-2-1 Dmc1催化股交換反應中的布朗運動變化 21 3-2-2 以自相關函數方式分析布朗運動-時間趨勢圖 23 第四章 總結與未來展望 27 4-1 論文總結 27 4-2 未來展望 28 附錄.................................................................................................................................30 A 以自相關函數分析 Dmc1 成核實驗中之布朗運動隨變化趨勢 30 A-1 實驗結果與討論 30 A-2 於 1 mM Ca2+ 中之 Dmc1 成核實驗布朗運動變化趨勢圖 35 A-3 於 2.5 mM Ca2+ 中之 Dmc1 成核實驗布朗運動變化趨勢圖 38 A-4 於 4 mM Ca2+ 中之 Dmc1 成核實驗布朗運動變化趨勢圖 40 B 對離去股實驗的拴球布朗運動進行分析 44 B-1 2.5 mM Ca2+ 中之離去股實驗拴球分析 44 B-2 2.5 mM Ca2+ 中之離去股實驗拴球分析 49 C 單分子栓球上的免疫螢光實驗 56 D 輔助蛋白 Hop2-Mnd1 和 DNA 的結合情形測定 62 參考文獻 106 | |
| dc.language.iso | zh-TW | |
| dc.title | 以單分子方法探討人類Dmc1蛋白在進行DNA股交換時的核蛋白結構 | zh_TW |
| dc.title | Investigating Human Dmc1 Nucleoprotein Structure During DNA Strand Exchange Using Single Molecule Methods | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蕭傳鐙(Chwan-Deng Hsiao),冀宏源(Hung-Yuan Chi),范秀芳(Hsiu-Fang Fan) | |
| dc.subject.keyword | 同源重組,DNA 修復,Dmc1,單分子栓球實驗,自相關函數, | zh_TW |
| dc.subject.keyword | homologous recombination,DNA repair,Dmc1,tethered particle motion,autocorrelation, | en |
| dc.relation.page | 110 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-08-15 | |
| 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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