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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81955完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李弘文(Hung-Wen Li) | |
| dc.contributor.author | Chia-Hua Lu | en |
| dc.contributor.author | 呂家驊 | zh_TW |
| dc.date.accessioned | 2022-11-25T03:07:24Z | - |
| dc.date.available | 2023-08-22 | |
| dc.date.copyright | 2021-11-11 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-23 | |
| dc.identifier.citation | 1 Sauvageau, S. et al. Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments. Mol. Cell Biol. 25, 4377-4387 (2005). 2 Keeney, S., Giroux, C. N. Kleckner, N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375-384 (1997). 3 Sonoda, E. et al. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 17, 598-608 (1998). 4 Sung, P. Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nature Reviews Molecular Cell Biology 7, 739-750 (2006). 5 Heyer, W. D., Ehmsen, K. T. Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genetics. 44, 113-139 (2010). 6 Crickard, J. B. Greene, E. C. The biochemistry of early meiotic recombination intermediates. Cell Cycle 17, 2520-2530 (2018). 7 Bishop, D. K., Park, D., Xu, L. Kleckner, N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439-456 (1992). 8 Masson, J. Y. West, S. C. The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem. Sci. 26, 131-136 (2001). 9 Story, R. M., Bishop, D. K., Kleckner, N. Steitz, T. A. Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast. Science 259, 1892 (1993). 10 Lin, Z., Kong, H., Nei, M. Ma, H. Origins and evolution of the recA\RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer. Proceedings of the National Academy of Sciences 103, 10328 (2006). 11 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). 12 Brown, M. S., Grubb, J., Zhang, A., Rust, M. J. Bishop, D. K. Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break. PLOS Genetics 11, e1005653 (2016). 13 Busygina, V. et al. Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1. DNA Repair (Amst) 12, 707-712 (2013). 14 Wold, M. S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61-92 (1997). 15 Yates, L. A. et al. A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA. Nature Comm. 9, 5447 (2018). 16 Flynn, R. L. Zou, L. Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians. Crit. Rev. Biochem. Mol. Biol. 45, 266-275 (2010). 17 Fan, J. Pavletich, N. P. Structure and conformational change of a replication protein A heterotrimer bound to ssDNA. Genes. Dev. 26, 2337-2347 (2012). 18 Wang, Q. M. et al. Human replication protein A induces dynamic changes in single-stranded DNA and RNA structures. J. Biol. Chem. 294, 13915-13927 (2019). 19 Chan, Y. L., Zhang, A., Weissman, B. P. Bishop, D. K. RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination. Nucleic Acids Research 47, 747-761 (2019). 20 Ishikawa-Ankerhold, H. C., Ankerhold, R. Drummen, G. P. C. Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17, 4047-4132 (2012). 21 Liao, T.-W. Developing Single-Molecule Fluorescence Imaging Platform to Study Dissociation Dynamics of RAD51 Recombinase Filament, National Taiwan University, (2018). 22 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). 23 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). 24 Lan, W. H. et al. Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase. Proc. Natl. Acad. Sci. USA 117, 11257-11264 (2020). 25 Ma, C. J., Gibb, B., Kwon, Y., Sung, P. Greene, E. C. Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament. Nucleic Acids Research 45, 749-761 (2017). 26 Cui, J., Kaandorp, J. A., Sloot, P. M., Lloyd, C. M. Filatov, M. V. Calcium homeostasis and signaling in yeast cells and cardiac myocytes. FEMS Yeast Res 9, 1137-1147 (2009). 27 Bonilla, M. Cunningham, K. W. Calcium release and influx in yeast: TRPC and VGCC rule another kingdom. Sci STKE 2002, pe17 (2002). 28 Cui, J. et al. Simulating calcium influx and free calcium concentrations in yeast. Cell Calcium 45, 123-132 (2009). 29 Cui, J. Kaandorp, J. A. Mathematical modeling of calcium homeostasis in yeast cells. Cell Calcium 39, 337-348 (2006). 30 Stathopoulos-Gerontides, A., Guo, J. J. Cyert, M. S. Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. Genes Dev 13, 798-803 (1999). 31 Montero, M. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 14, 5467-5475 (1995). 32 Hindi, S. M., Tajrishi, M. M. Kumar, A. Signaling Mechanisms in Mammalian Myoblast Fusion. Science Signaling 6, re2 (2013). 33 Przybylski, R. J., Szigeti, V., Davidheiser, S. Kirby, A. C. Calcium regulation of skeletal myogenesis. II. Extracellular and cell surface effects. Cell Calcium 15, 132-142 (1994). 34 Reitz, D., Grubb, J. Bishop, D. K. A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability. PLOS Genetics 15, e1008217 (2019). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81955 | - |
| dc.description.abstract | 同源重組反應為細胞修復 DNA 雙股斷裂時主要的方式之一，而此反應中的第一步驟為同源重組酶組裝到受損的單股 DNA 形成穩定的核蛋白絲。在釀酒酵母菌 (Saccharomyces cerevisiae) 的 ScDmc1 可以有效的被 Ca2+ 調控以提高其股交換反應，及抑制其水解 ATP 的能力。我們利用單分子螢光技術發現，在不需要其他輔助蛋白的幫助下，Ca2+ 可以刺激 ScDmc1 組裝到已被 ScRPA 結合的單股 DNA 上，然而此刺激效果並沒有發生在真核生物中另一種同源重組酶 ScRad51。此外，Ca2+ 刺激 ScDmc1 取代 ScRPA 只發生在低鹽(50 mM KCl)條件下，其中的原因為 ScDmc1 在 Ca2+ 存在下，對於只有 9 個鹼基的短 DNA，其結合速率在低鹽條件下與高鹽(150 mM KCl)條件相比較為快速。最後，我們深入探討 ScDmc1 是如何開始成核在被 ScRPA 預先結合的單股上並提出三個可能模型，其一為 ScDmc1 首先成核在雙股並延伸核蛋白絲取代 ScRPA，另一則是成核在單股 3' 尾端並延伸核蛋白絲，最後則是成核在 5' 單/雙股交界處，我們利用螢光分子對修飾在 DNA 基質的不同位置去間接剔除前兩種模型的可能性，並確認了 ScDmc1 在組裝到被 ScRPA 預先結合的單股 DNA 上也對5' 單/雙股交界處展現偏好性。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T03:07:24Z (GMT). No. of bitstreams: 1 U0001-2308202114474700.pdf: 5131434 bytes, checksum: 46360dc0f3e31438ba1b800809239873 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 誌謝 ii 摘要 iv Abstract v 目錄 vi 圖目錄 viii 表目錄 ix 第一章 緒論 1 1-1 同源重組反應 (Homologous recombination) 1 1-2 Dmc1 與 Rad51 蛋白 2 1-3 RPA 3 1-4 Ca2+ 對ScDmc1 之刺激 4 1-5 研究動機 6 第二章 實驗方法與設計 7 2-1 蛋白質來源 7 2-2 DNA 基質設計 8 2-3 溶液配方與製備 10 2-4 反應玻片製備 11 2-5 單分子螢光共振能量轉移 (smFRET) 理論 13 2-6 單分子螢光光路架設及顯微鏡 14 2-7 單分子螢光共振能量轉移實驗流程 15 2-8 單分子螢光共振能量轉移數據分析 15 2-9 Thermal Shift Assay 17 第三章 實驗結果與討論 18 3-1 Ca2+ 刺激 ScDmc1 取代預先結合到單股上的 ScRPA 18 3-2 抑制 ATP 水解無法刺激 ScDmc1 組裝到 RPA-coated 之單股 DNA 21 3-3 Ca2+ 濃度對 ScDmc1 取代 ScRPA 之效率影響 23 3-4 Ca2+ 對 ScDmc1 構型之改變 25 3-5 高鉀條件下 ScDmc1 取代 ScRPA 之效率與低鉀條件相比急遽下降，其因為ScDmc1 結合到較短的 ssDNA 速率與低鉀條件相比下降。 26 3-6 Ca2+ 刺激 ScDmc1 成核在單/雙股交界處並延長核蛋白絲取代 ScRPA 之機制 30 第四章 實驗總結與未來展望 34 4-1 實驗總結 34 4-2 未來展望 37 參考文獻 39 | |
| dc.language.iso | zh-TW | |
| dc.subject | 5'單/雙股交界處 | zh_TW |
| dc.subject | Ca2+ | zh_TW |
| dc.subject | ScDmc1 | zh_TW |
| dc.subject | ScRad51 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | 5' junction | en |
| dc.subject | Ca2+ | en |
| dc.subject | ScRad51 | en |
| dc.subject | ScDmc1 | en |
| dc.subject | Homologous recombination | en |
| dc.title | Ca2+ 刺激 ScDmc1 組裝至 ScRPA 結合之 DNA 的機制探究 | zh_TW |
| dc.title | Mechanistic Studies on How Ca2+ Stimulates the Assembly of Yeast Dmc1 on RPA-coated DNA | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 冀宏源(Hsin-Tsai Liu),林敬哲(Chih-Yang Tseng),王廷方 | |
| dc.subject.keyword | 同源重組,ScDmc1,ScRad51,Ca2+,5'單/雙股交界處, | zh_TW |
| dc.subject.keyword | Homologous recombination,ScDmc1,ScRad51,Ca2+,5' junction, | en |
| dc.relation.page | 48 | |
| dc.identifier.doi | 10.6342/NTU202102622 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-25 | |
| dc.contributor.author-college | 理學院 | zh_TW |
| dc.contributor.author-dept | 化學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-23 | - |
| 顯示於系所單位： | 化學系 | |
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