臺灣博碩士論文加值系統

當原核生物在複製去氧核醣核酸的過程中，發生雙股去氧核醣核酸斷裂時，生物體會啟動同源重組修復的途徑，而RecA蛋白參與其中並扮演重要的角色， RecA蛋白會結合到單股去氧核醣核酸上，形成核蛋白絲，接著尋找同源互補的序列，最後進行股交換。Cox團隊先前研究發現大腸桿菌RecA的 C端少了末17個胺基酸相較於大腸桿菌野生型RecA 對於單股去氧核醣核酸具有較高的結合力，而股交換反應偏好的環境也從酸性轉換為鹼性。先前已知就算遭遇數百個雙股去氧核醣核酸的斷裂，抗輻射奇異球菌RecA也可以在幾小時內修復，而其股交換的活性偏好於鹼性環境。奇異球菌屬RecA的C端末端 (第280~352號胺基酸) 胺基酸擁有的負電多數少於大腸桿菌 RecA，為了探討RecA的C端末端 (第280~352號胺基酸) 對於去氧核醣核酸股交換的影響，我們將菩提奇異球菌 C端末17個胺基酸替換成大腸桿菌RecA的序列 (菩提奇異球菌-C17E. coli RecA)，藉由凝膠阻滯分析實驗觀察不同物種的RecA 股交換偏好的pH值，透過單分子栓球實驗觀察核蛋白絲形成的速率，最後利用時間解析度較高的單分子螢光共振能量轉移實驗觀察RecA 核蛋白絲形成的穩定性和動態資訊。

RecA protein is essential for repairing the damaged chromosomal DNA by mediating homologous recombination. RecA can bind on ssDNA to form a nucleoprotein filament. Later, a RecA nucleoprotein filament can search for homologous sequence from another duplex dsDNA. The last step is the RecA dissociation after the completion of strand exchange. Previously, Cox et al. reported that a strain expressing C-terminal truncated RecA protein does not exhibit more UV-sensitive than a wild type strain. RecA ΔC17 possesses a higher ssDNA binding affinity than the wt RecA. Moreover, the deletion of residues in the C-terminus of RecA will shift the pH reaction profile of strand exchange to a high pH condition. The C-terminus of most RecA proteins from Deinococcus genus is shorter and lack of negative-charged residues than that of E. coli RecA. D. ficus RecA, which has 92% sequence identity to Dr. RecA, was found to possess a similar ATPase activity and a similar DNA strand exchange reaction pathway to those of E. coli RecA. The pH profile of DNA strand exchange efficiency is shifted toward higher pH for D. ficus RecA. Here, we replaced the last 17 amino acid of D. ficus C-terminus with that of E. coli RecA to construct a chimera protein, D. ficus- C17E. coli RecA. Previously, we found that the rate-limited step is nucleoprotein formation in the strand exchange process for E. coli RecA and D. ficus-C17E. coli RecA with TPM experiment. In contrast, D. ficus RecA and E. coli ΔC17 RecA possess higher DNA binding affinities and lower protonation requirements for the formation of nucleoprotein filament. In order to investigate the influence of C-terminal residues in the formation of nucleoprotein filament, we would like to apply FRET experiment to observe dynamic and stability of RecA nucleoprotein filament in detail.

中英對照表..........................i
中文摘要............................iii
英文摘要............................iv
目錄................................v
圖目錄..............................viii
表目錄..............................ix
第一章 緒論.........................1
1-1 RecA 蛋白的結構..................1
1-2 RecA 蛋白的功能..................1
1-3 RecA 蛋白C端末端 (第280~352號胺基酸)的重要性...2
1-4 研究動機.........................4
1-5實驗流程..........................4
第二章 實驗材料製備和實驗設計............6
2-1 去氧核醣核酸製備.....................6
2-1.1 去氧核醣核酸(凝膠阻滯分析實驗用)...........6
2-1.2 去氧核醣核酸(單分子栓球實驗用).............6
2-1.3 去氧核醣核酸(單分子螢光共振能量轉移實驗用)...6
2-2 萃取菩提奇異球菌和菩提奇異球菌-C17E.coli RecA質體.......7
2-3 蛋白質純化..........................7
2-4 股交換實驗過程.......................10
2-5 單分子拴球反應小球修飾................10
2-6 聚乙二醇反應玻片製備..................11
2-7 單分子拴球實驗過程....................12
2-7.1 數據分析........................12
2-8 單分子螢光共振能量轉移實驗儀器架設......13
2-8.1 光學顯微鏡架設...................13
2-8.2 校正Cy3參數.....................13
2-9 單分子螢光共振能量轉移實驗過程.........14
2-9.1 除氧系統........................15
2-9.2 數據分析........................15
第三章 實驗結果與討論....................17
3-1 蛋白質純化...........................17
3-2 股交換活性實驗.......................17
3-3 單分子拴球實驗.......................18
3-4 單分子螢光共振能量轉移實驗............20
第四章 實驗結論與未來展望................25
圖...............................27
表...............................47
參考文獻.........................49
附錄.............................53
附錄一、參考文獻圖...................53
附錄二、實驗使用之藥品清單................67
附錄三、實驗使用之引子清單...................70
附錄四、實驗使用之溶液清單....................71
附錄五、菩提奇異球菌RecA和菩提奇異球菌-C17E.coli RecA 胺基酸定序...75

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