臺灣博碩士論文加值系統

無論是古細菌、原核或是真核生物的同源重組蛋白，皆在電子顯微鏡下觀察到6-8個單元形成的閉環多聚體，但是實際上在水溶液狀態中更直接的結構資訊則始終不足夠，本實驗利用單分子光鉗以直接的方式量測mRAD51多聚體是否真實存在，且以此單元直接組裝到DNA上。為了精進光鉗系統之穩定度及量測準確度，使的系統解析度足以觀察此單元的即時組裝過程，我們對系統做了以下改進； 1. 將原本以電腦為運算基底的雷射回饋系統改為用電路回饋，有效提升了約一數量級的雷射穩定度。 2. 使用三種獨立的校正方法，準確地決定出光鉗的彈力常數。 3. 以被動式定力鉗取代先前主動式定力鉗的實驗架設，使系統複雜度降低，及提高量測的空間及時間解析度。 4. 利用Allan deviation分析被動式定力鉗穩定度，在46.5 pN情況下，使用一條包含4178 bp雙股和1002 nt單股的DNA，可以在100 ms下得到1 nm的解析度。我們利用被動式定力鉗 (passive force clamp assay)高解析度的直接量測mRAD51與DNA結合過程；在組裝的過程中，可觀察到不連續的、隨機的長度停滯再變長現象，而前後兩個停滯狀態的長度變化，大部分對應到8個mRAD51與DNA的結合。這個結果直接證明了mRAD51與DNA結合的主要單元為8個mRAD51。當加入輔助蛋白mSWI5-SFR1聚合體，結合大小變為以4個mRAD51的結合單元，同時發現核蛋白絲生成速率變為原本之3倍。在加入輔助蛋白mBRCA2之胜肽片段mBRC4後，也降低結合的單元到4個mRAD51並提高核蛋白絲生成速率為原本之5倍。我們的研究顯示，SWI5-SFR1與BRC4可能是以減少RAD51聚合體的大小以及改變RAD51的構型以增加對DNA親和力的方式促進RAD51核蛋白絲的生成。

Recombinases are shown to form closed-ring (hexameric to octameric) structure in electron microscopic studies, but direct structural evidence in solution is missing. We used the passive force-clamp optical tweezers to directly observe the existence of oligomeric recombinase nucleoprotein filament assembly in real-time. To achieve sufficient resolution to resolve individual steps during filament assembly, several instrumental improvements and programming developments have been achieved: (1) improved our home-built optical tweezers stability by using a circuit-based laser feedback to reduce laser power fluctuation; (2) improved the precision and accuracy of trap stiffness calibration by using three independent methods; (3) developed an automatical program to implement passive force cl (4) used Allan deviation to validate passive force clamp. Our improved instrument can achieve 1 nm precision in 100 ms using force-activated DNA (a DNA contain 4178 bp dsDNA and 1002 nt ssDNA). We used this improved passive force-clamp optical tweezers to directly observe the assembly dynamics of mouse RAD51 nucleoprotein filament in real-time. Stepwise extension is observed with a single-exponentially distributed dwell between continous extension. Analysis showed that mRAD51 mostly assemble in octamers. Adding accessory proteins, mSWI5-SFR1 complex reduces the assembly unit from octamers to tetramers. Including a short peptide of BRCA2 protein, BRC4, also reduces to tetramers. This reduction in assembly unit is accompanied by increase in extension rates. Our results suggest that the stimulation of mRAD51 filament assembly by mSWI5-SFR1 and mBRC4 are resulted from reduing the assembly unit and increasing the affinity of mRAD51 to DNA.

摘要 I
ABSTRACT II
目錄 III
圖目錄 vi
表目錄 ix
第一章 緒論 - 1 -
1.1 同源重組反應 - 1 -
1.2 研究動機 - 1 -
輔助蛋白SWI5-SFR1對RAD51之影響 - 6 -
輔助蛋白BRCA2之小片段胜肽BRC4對RAD51之影響 - 7 -
第二章 實驗方法與設計 - 8 -
2.1 mRAD51、mSWI5-SFR1、mBRC4蛋白的純化與保存 - 8 -
2.2 DNA基質合成 - 8 -
3281 bp dsDNA - 8 -
5180 bp 力活化單股DNA (force-activated DNA) - 9 -
20 bp 髮夾型DNA - 11 -
2.3 實驗操作方法 - 12 -
azide-PEG玻片之表面修飾 - 13 -
製備表面修飾卵白素之聚苯乙烯球 - 13 -
製備反應用微流道 - 13 -
實驗用緩衝溶液 - 13 -
第三章 光鉗系統架設與校正 - 14 -
3.1 光鉗原理 - 14 -
3.2 光路設計 - 15 -
聲光調變器AOM (acousto-optical modulator ) - 17 -
四象限光電感測器QPD (quadrant photodiode) - 18 -
光鉗系統儀器單元 - 20 -
3.3 雷射功率穩定系統 - 21 -
3.4 DNA長度之幾何校正 - 25 -
DNA栓球高度決定 - 26 -
3.5 光鉗雷射之力常數校正 - 28 -
以能量均分原理 (equipartition theorem, EQP)校正雷射彈力常數 - 29 -
以流體力學 (hydrodynamics method, HYD)校正雷射彈力常數 - 29 -
以能量密度頻譜分析 (power spectral density, PSD)校正雷射彈力常數 - 31 -
3.6 光鉗位置偵測系統校正 - 36 -
固定小球掃描 (stuck-bead scan, SBC) - 36 -
主動式能量密度頻譜 - 38 -
3.7 光鉗校正步驟 - 42 -
3.8 力與伸長量曲線 (force-extension curve, FEC) - 43 -
3.9 量測DNA力學性質 - 45 -
Marko-Siggia worm-like chain (M-S WLC) - 45 -
Odijk extensible worm-like chain (eWLC) - 45 -
Smith extensible freely-jointed chain (eFJC) - 46 -
Modified worm-like chain (mWLC) - 46 -
雙股DNA力學性質統計 - 49 -
單股髮夾形二級結構分析來確認光鉗系統校正 - 50 -
力活化單股DNA分析 (force-activated DNA) - 52 -
3.10 被動式定力鉗實驗 (passive force clamp assay) - 55 -
定力校正 - 57 -
靈敏度校正 - 59 -
3.11 系統穩定度 - 60 -
以Allan Deviation決定系統穩定度 - 60 -
被動式定力鉗－單分子栓球穩定度 - 61 -
第四章 實驗結果與討論 - 65 -
4.1 mRAD51-DNA核蛋白絲之力學性質 - 65 -
4.2 mRAD51組裝在活化的力引發單股DNA - 68 -
4.3 被動式定力鉗之高解析度時間軌跡分析 - 70 -
mRAD51之結合大小、步伐停留時間分析 - 72 -
mRAD51+mSWI5-SFR1之結合步伐、步伐停留時間分析 - 79 -
mRAD51+mBRC4 peptide之結合步伐、步伐停留時間分析 - 81 -
比較三組數據 - 83 -
第五章 總結與未來展望 - 89 -
參考文獻 - 91 -
附錄 - 93 -
附錄一 DNA引子序列 - 93 -
附錄二 製備表面修飾卵白素(streptavidin) 的直徑1030 奈米聚苯乙烯球 - 94 -
附錄三 被動式定力鉗時間軌跡圖 - 95 -

1.Yu, X.; Egelman, E. H., The RecA hexamer is a structural homologue of ring helicases. Nat. Struct. Biol. 1997, 4 (2), 101-104.
2.Yang, S., et al., Archaeal RadA protein binds DNA as both helical filaments and octameric rings11Edited by M. Belfort. J. Mol. Biol. 2001, 314 (5), 1077-1085.
3.Shin, D. S., et al., Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J. 2003, 22 (17), 4566-4576.
4.Passy, S. I., et al., Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (19), 10684-10688.
5.van der Heijden, T., et al., Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules. Nucleic Acids Res. 2007, 35 (17), 5646-5657.
6.Hilario, J., et al., Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (2), 361.
7.Su, G.-C., et al., Role of the RAD51–SWI5–SFR1 Ensemble in homologous recombination. Nucleic Acids Res. 2016, 44 (13), 6242-6251.
8.Kokabu, Y., et al., Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure. Journal of Biological Chemistry 2011, 286 (50), 43569-43576.
9.Tsai, S.-P., et al., Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex. Nucleic Acids Res. 2012, 40 (14), 6558-6569.
10.Lu, C.-H., et al., Swi5–Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation. Proc. Natl. Acad. Sci. U.S.A. 2018, 115 (43), E10059.
11.Carreira, A.; Kowalczykowski, S. C., Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (26), 10448.
12.Davies, A. A., et al., Role of BRCA2 in Control of the RAD51 Recombination and DNA Repair Protein. Molecular Cell 2001, 7 (2), 273-282.
13.Su, G.-C., et al., Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex. Nucleic Acids Res. 2014, 42 (1), 349-358.
14.Okoniewski, S. R.; Uyetake, L.; Perkins, T. T., Force-activated DNA substrates for probing individual proteins interacting with single-stranded DNA. Nucleic Acids Res 2017, 45 (18), 10775-10782.
15.Woodside, M. T., et al., Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (16), 6190.
16.Eeftens, J. M., et al., Copper-free click chemistry for attachment of biomolecules in magnetic tweezers. BMC Biophys. 2015, 8 (1), 9.
17.Walder, R., et al., Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation. J. Am. Chem. Soc. 2017, 139 (29), 9867-9875.
18.Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. 2001, 40 (11), 2004-2021.
19.Neuman, K. C.; Block, S. M., Optical trapping. Rev. Sci. Instrum. 2004, 75 (9), 2787-2809.
20.Okoniewski, S. R.; Carter, A. R.; Perkins, T. T., A Surface-Coupled Optical Trap with 1-bp Precision via Active Stabilization. Methods Mol Biol 2017, 1486, 77-107.
21.Wang, M. D., et al., Stretching DNA with optical tweezers. Biophys. J. 1997, 72 (3), 1335-1346.
22.Neuman, K. C.; Abbondanzieri, E. A.; Block, S. M., Measurement of the effective focal shift in an optical trap. Opt. Lett. 2005, 30 (11), 1318-1320.
23.Perkins, T. T., Optical traps for single molecule biophysics: a primer. Laser Photonics Rev. 2009, 3 (1-2), 203-220.
24.Svoboda, K.; Block, S. M., Biological Applications of Optical Forces. Annu. Rev. Biophys. 1994, 23 (1), 247-285.
25.Berg-Sørensen, K.; Flyvbjerg, H., Power spectrum analysis for optical tweezers. Rev. Sci. Instrum. 2004, 75 (3), 594-612.
26.Tolić-Nørrelykke, S. F., et al., Calibration of optical tweezers with positional detection in the back focal plane. Rev. Sci. Instrum. 2006, 77 (10), 103101.
27.Bustamante, C., et al., Entropic elasticity of lambda-phage DNA. Science 1994, 265 (5178), 1599.
28.Marko, J. F.; Siggia, E. D., Stretching DNA. Macromolecules 1995, 28 (26), 8759-8770.
29.Odijk, T., Stiff Chains and Filaments under Tension. Macromolecules 1995, 28 (20), 7016-7018.
30.Smith, S. B.; Cui, Y.; Bustamante, C., Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules. Science 1996, 271 (5250), 795.
31.Cocco, S., et al., Overstretching and force-driven strand separation of double-helix DNA. Phys. Rev. E 2004, 70 (1), 011910.
32.van Mameren, J., et al., Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (43), 18231.
33.Zhang, X., et al., Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (10), 3865.
34.Greenleaf, W. J., et al., Passive All-Optical Force Clamp for High-Resolution Laser Trapping. Phys. Rev. Lett. 2005, 95 (20), 208102.
35.Czerwinski, F.; Richardson, A. C.; Oddershede, L. B., Quantifying Noise in Optical Tweezers by Allan Variance. Opt. Express 2009, 17 (15), 13255-13269.
36.Perkins, T. T., Ångström-Precision Optical Traps and Applications. Annu. Rev. Biophys. 2014, 43 (1), 279-302.
37.Hegner, M.; Smith, S. B.; Bustamante, C., Polymerization and mechanical properties of single RecA–DNA filaments. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (18), 10109.
38.Ogawa, T., et al., Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 1993, 259 (5103), 1896.
39.Abbondanzieri, E. A., et al., Direct observation of base-pair stepping by RNA polymerase. Nature 2005, 438 (7067), 460-465.
40.Carreira, A., et al., The BRC Repeats of BRCA2 Modulate the DNA-Binding Selectivity of RAD51. Cell 2009, 136 (6), 1032-1043.