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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李弘文 | |
dc.contributor.author | CHIH-HAO LU | en |
dc.contributor.author | 盧致豪 | zh_TW |
dc.date.accessioned | 2021-07-11T14:38:12Z | - |
dc.date.available | 2022-07-31 | |
dc.date.copyright | 2017-07-31 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-07-27 | |
dc.identifier.citation | REFERENCES
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Rad51 Paralog Complexes BCDX2 and CX3 Act at Different Stages in the BRCA1-BRCA2-Dependent Homologous Recombination Pathway. Mol Cell Biol 33, 387-395 (2013). 8 Takata, M. et al. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol 21, 2858-2866 (2001). 9 Sugawara, N., Wang, X. & Haber, J. E. In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell 12, 209-219 (2003). 10 Kokabu, Y. et al. Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure. J Biol Chem 286, 43569-43576 (2011). 11 Liu, J. et al. Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation. Nature 479, 245-U129 (2011). 12 Haruta, N. et al. The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro. Nat Struct Mol Biol 13, 823-830 (2006). 13 Liu, J. & Heyer, W. D. Who's who in human recombination: BRCA2 and RAD52. P Natl Acad Sci USA 108, 441-442 (2011). 14 Zhao, W. X. et al. Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry. Mol Cell 59, 176-187 (2015). 15 Esashi, F., Galkin, V. E., Yu, X., Egelman, E. H. & West, S. C. Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat Struct Mol Biol 14, 468-474 (2007). 16 Kuwabara, N. et al. Mechanistic Insights into the Activation of Rad51-Mediated Strand Exchange from the Structure of a Recombination Activator, the Swi5-Sfr1 Complex. Structure 20, 440-449 (2012). 17 Kurokawa, Y., Murayama, Y., Haruta-Takahashi, N., Urabe, I. & Iwasaki, H. Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators. Plos Biol 6, 836-848 (2008). 18 Su, G. C. et al. Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex. Nucleic Acids Res 42, 349-358 (2014). 19 Su, G. C. et al. Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination. Nucleic Acids Res 44, 6242-6251 (2016). 20 Tsai, S. P. et al. Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex. Nucleic Acids Res 40, 6558-6569 (2012). 21 Yang, H. J., Li, Q. B., Fan, J., Holloman, W. K. & Pavletich, N. P. The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction. Nature 433, 653-657 (2005). 22 Davies, O. R. & Pellegrini, L. Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats. Nat Struct Mol Biol 14, 475-483 (2007). 23 Lee, M., Lipfert, J., Sanchez, H., Wyman, C. & Dekker, N. H. Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament. Nucleic Acids Res 41, 7023-+ (2013). 24 Ristic, D. et al. Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function. Nucleic Acids Res 33, 3292-3302 (2005). 25 Mine, J. et al. Real-time measurements of the nucleation, growth and dissociation of single Rad51-DNA nucleoprotein filaments. Nucleic Acids Res 35, 7171-7187 (2007). 26 Candelli, A., Modesti, M., Peterman, E. J. G. & Wuite, G. J. L. Single-molecule views on homologous recombination. Q Rev Biophys 46, 323-348 (2013). 27 Candelli, A. et al. Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution. P Natl Acad Sci USA 111, 15090-15095 (2014). 28 Hilario, J., Amitani, I., Baskin, R. J. & Kowalczykowski, S. C. Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. P Natl Acad Sci USA 106, 361-368 (2009). 29 Gal, J., Schnell, R., Szekeres, S. & Kalman, M. Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR). Mol Gen Genet 260, 569-573 (1999). 30 Kuwabara, N. et al. Expression, purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast. Acta Crystallogr F 66, 1124-1126 (2010). 31 Chung, C. & Li, H. W. Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases. J Am Chem Soc 135, 8920-8925 (2013). 32 Lu, C. H., Chang, T. T., Cho, C. C., Lin, H. C. & Li, H. W. Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity. unpublished work. 33 Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515-527 (2006). 34 Lu, Y. W. et al. Using Single-Molecule Approaches To Study Archaeal DNA-Binding Protein Alba1. Biochemistry-Us 52, 7714-7722 (2013). 35 Piechura, J. R. et al. Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation. DNA Repair 26, 30-43 (2015). 36 Liu, Y. L., Stasiak, A. Z., Mcllwraith, M. J., Stasiak, A. & West, S. C. Conformational changes modulate the activity of human RAD51 protein. J Mol Biol 337, 817-827 (2004). 37 Wu, H. Y., Lu, C. H. & Li, H. W. RecA Nucleoprotein Filament Formation on SSB-wrapped DNA Includes RecA-SSB Interaction. unpublished work. 38 Shinohara, A. et al. Cloning of Human, Mouse and Fission Yeast Recombination Genes Homologous to Rad51 and Reca (Vol 4, Pg 239, 1993). Nat Genet 5, 312-312 (1993). 39 Cartwright, R., Dunn, A. M., Simpson, P. J., Tambini, C. E. & Thacker, J. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family. Nucleic Acids Res 26, 1653-1659 (1998). 40 Shin, D. S. et al. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. Embo J 22, 4566-4576 (2003). 41 Lusetti, S. L. et al. The RecF protein antagonizes RecX function via direct interaction. Mol Cell 21, 41-50 (2006). 42 Candelli, A. et al. Resolving RAD51 Filament Nucleation, Extension and Disassembly on ssDNA and dsDNA One Protein at a Time. Biophys J 100, 240-240 (2011). 43 Saikusa, K. et al. Characterisation of an intrinsically disordered protein complex of Swi5-Sfr1 by ion mobility mass spectrometry and small-angle X-ray scattering. Analyst 138, 1441-1449 (2013). 44 Ferrari, S. R., Grubb, J. & Bishop, D. K. The Mei5-Sae3 Protein Complex Mediates Dmc1 Activity in Saccharomyces cerevisiae. J Biol Chem 284, 11766-11770 (2009). 45 Hayase, A. et al. A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1. Cell 119, 927-940 (2004). 46 Kim, S. H. et al. Cooperative Conformational Transitions Keep RecA Filament Active During ATPase Cycle. J Am Chem Soc 136, 14796-14800 (2014). 47 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). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77954 | - |
dc.description.abstract | 真核生物中的RAD51蛋白是驅動DNA同源重組修護反應最重要的重組酵素(recombinase);RAD51會結合到受損、裸露的單股DNA上形成核蛋白絲(nucleoprotein filament),形成RAD51-單股DNA的核蛋白絲是催化後續同源配對(homology pairing)以及股交換反應(strand exchange)的必要步驟。RAD51核蛋白絲生成反應主要可以區分為較慢的成核反應(nucleation)以及非常快速的延伸反應(extension);為了克服成核反應的高能量障壁,有許多輔助蛋白(accessory proteins)參與並加速RAD51的成核反應,以增加整體DNA同源重組修護反應的效率,其中SWI5-SFR1輔助蛋白(S5S1)被證明擁有和RAD51形成複合體(complex)、刺激RAD51所調節的DNA同源重組反應。在此篇論文中,我們利用單分子栓球實驗(single-molecule tethered particle motion, smTPM)以及單分子螢光共振能量轉移實驗(single-molecule Forster resonance energy transfer, smFRET)證明老鼠SWI5-SFR1能有效地刺激老鼠RAD51的成核反應。我們同時也證明這樣的刺激功能主要由以下幾種機制來達成:(一)減少RAD51成核單位 (nucleation unit); (二)增加RAD51對單股DNA的親和力(ssDNA affinity) ; (三)穩定RAD51所形成的核 (nuclei);然而SWI5-SFR1在另一個物種-分裂生殖酵母菌(Schizosaccharomyces pombe, Sp)-並沒有刺激成核反應的功能,但是酵母菌SWI5-SFR1擁有防止RAD51核蛋白絲解離(disassembly)的功能,且其效率比起老鼠SWI5-SFR1還要高。在不同的物種中,即使是同一種輔助蛋白,它們也會因應RAD51在物種間不同的性質和活性而有不同的調節功能:老鼠SWI5-SFR1有效地刺激老鼠RAD51的成核反應,而酵母菌SWI5-SFR1擁有防止RAD51核蛋白絲解離的功能,但最後都能達到穩定RAD51核蛋白絲的效果。 | zh_TW |
dc.description.abstract | Eukaryotic RAD51 protein is essential for DNA homologous recombination in repairing doule-stranded DNA breaks. RAD51 recombinases assemble onto ssDNA to form a presynaptic filament, the required functional component for homology pairing and strand exchange reactions. This filament assembly is the committed step of homologous recombination and is subjected to regulation. Nucleation step is kinetically slow, and several accessory proteins have been identified to regulate RAD51 nucleation. SWI5-SFR1 (S5S1) is a heterodimeric accessory protein, and previous biochemical work showed that S5S1 interacts with RAD51, and stimulates RAD51-mediated homologous recombination. Our single-molecule tethered particle motion (TPM) and single-molecule fluorescence resonance energy transfer (smFRET) experiments demonstrated that mouse S5S1 interacts with mouse RAD51 to form complex, and the mRAD51-S5S1 complex efficiently stimulates the nucleation step. We also showed that mS5S1 stimulates mRAD51 nucleation by (i) reducing mRAD51 seed size, (ii) increasing mRAD51 ssDNA affinity and (iii) stabilizing mRAD51 nucleus on ssDNA. While nucleation stimulation by S5S1 is absent in fission yeast (Schizosaccharomyce pombe, Sp) system in our single-molecule work, SpS5S1 is shown to prevent SpRad51 disassembly. Different regulation strategies among species allow S5S1 to stabilize Rad51 filament efficiently. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:38:12Z (GMT). No. of bitstreams: 1 ntu-106-R04223149-1.pdf: 3650095 bytes, checksum: 132114048bf9f34061d8082fe13b594f (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | TABLE OF CONTENTS
謝辭 i 摘要 iii Abstract iv LIST OF FIGURES viii LIST OF TABLES x CHAPTER 1: Introduction 1 1-1 RAD51-mediated homologous recombination and associated accessory protein - SWI5-SFR1 1 1-2 Motivation 7 CHAPTER 2: Materials and Methods 10 2-1 Proteins 10 2-2 DNA substrate prepartion 10 2-2.1 Gapped DNA substrate preparation (For TPM assembly and disassembly experiments) 10 2-2.2 Dye-labeled hydrid DNA substrate preparation (For smFRET experiments) 14 2-3 Buffer condition 15 2-3.1 1X Mouse RAD51 reaction buffer (For TPM assembly and disassembly experiments) 15 2-3.2 1X S. pombe Rad51 reaction buffer (For TPM assembly and disassembly experiments) 15 2-3.3 Oxygen scavenging system (Imaging buffer, For mouse RAD51 smFRET experiments) 15 2-3.4 1X annealing buffer 16 2-3.5 1X T50 buffer (For streptaviding removal and DNA subtrate serial dilution in smFRET experiments) 16 2-4 Reaction chamber preparation 16 2-4.1 Ordinary glass slide preparation (For Single-molecule TPM assembly experiment) 16 2-4.2 Silanized glass slide preparation (For Single-molecule TPM disassembly experiment) 16 2-4.3 PEGylated glass slide preparation (For Single-molecule FRET experiment) 17 2-5 Experimental Procedure 18 2-5.1 Single-molecule tethered particle motion (TPM) 18 2-5.2 Single-molecule TPM assembly experiment (Figure 2-5) 19 2-5.3 Single-molecule TPM disassembly experiment (Figure 2-6) 20 2-5.4 Single-molecule fluorescence resonance energy transfer (smFRET) experiment 20 2-6 Data Analysis 21 2-6.1 Single-molecule TPM assembly and disassembly experiment 21 2-6.2 Single-molecule FRET experiment 25 CHAPTER 3: Results 27 3-1 Mouse SWI5-SFR1 (S5S1) stimulates mRAD51 nucleoprotein filament assembly 27 3-2 mS5S1 stimulation on mRAD51 nucleation step depends on mS5S1 concentrations 32 3-3 mS5S1 stimulates mRAD51 assembly onto ssDNA by reducing mRAD51 nucleation unit 38 3-4 mS5S1 stabilizes mRAD51 nucleus on ssDNA 43 3-5 mS5S1 stimulates mRAD51 assembly onto ssDNA by increasing mRAD51 ssDNA affinity 50 3-6 Higher Schizosaccharomyces pombe (Sp) S5S1 concentrations inhibit SpRad51 filament assembly 55 3-7 SpS5S1 prevent Rad 51 filament disassembly more efficiently than mS5S1 66 CHAPTER 4: Discussion, Conclusion and Future perspective 73 4-1 Discussion 73 4-2 Conclusion 80 4-3 Futrure perspective 80 REFERENCES 82 Appendix 86 I. List for enzymmes, chemicals and vendors 86 II. List for primers 89 III. Calculation of mRAD51 fractional occupancy in mRAD51-S5S12 complex 90 | |
dc.language.iso | en | |
dc.title | 利用單分子技術探討SWI5-SFR1促進DNA同源重組酶-RAD51核蛋白絲形成的機制 | zh_TW |
dc.title | Single-molecule biochemical studies on the stimulation mechanism of SWI5-SFR1 complex on RAD51 presynaptic filament formation | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳昭岑,林敬哲,冀宏源,范秀芳,王廷方 | |
dc.subject.keyword | RAD51,SWI5-SFR1,DNA同源重組修護,單分子栓球實驗,單分子螢光共振能量轉移, | zh_TW |
dc.subject.keyword | RAD51,SWI5-SFR1,DNA homologous recombinational repair,smTPM,smFRET, | en |
dc.relation.page | 90 | |
dc.identifier.doi | 10.6342/NTU201702131 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-07-27 | |
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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