臺灣博碩士論文加值系統

2006年發現的新物種Deincoccus ficus (D.ficus)，隸屬於Deinococcus 奇異球菌屬，此屬菌株具有高劑量UV的耐受度，而其中Deinococcus radiodurain (Dr)抗輻射奇異球菌屬已被廣泛研究，且得知其能承受大量的雙股DNA雙股斷裂。Dr與D.ficus間基因序列的相似度相當高，然而D.ficus被發現能生長於較鹼性的環境中，具備有Dr不會發生的UV-誘導突變，因此認為D.ficus 具有特殊的DNA修補系統。而作為DNA修補反應中的主要蛋白，RecA被選為研究的主要對象。我們首先觀察D.ficus RecA進行股交換反應的偏好性，發現於偏鹼環境中具有較高的活性，然而此特性與核酸-蛋白纖維的穩定度並不同步，且此活性偏好與一般大腸桿菌的RecA不同，因此進一步使用單分子系統-栓球實驗-觀察反應的細部活性。再觀測過核酸-蛋白纖維和三股中間形階段產物的穩定度、計算同源搜尋以及DNA置換的發生頻率後，發現導致D.ficus的RecA異於大腸桿菌的主要原因為中間產物的穩定度差異以及速率決定步驟的不同。

Deinococcus genuses are strongly resistant to UV irradiant due to their high tolerance of double strand breaks DNA. It has been reported that Deinococcus radiodurain has robust DNA-damage repair system to resist the UV irradiation-caused DNA damage. A new species, Deincoccus ficus (D.ficus), was found in 2006. Both species have high similarities in genome sequence. However D. ficus has been found to survive under basic condition and mutation was found upon UV irradiation. Based on these findings, the hypothesis that DNA-damage repair system in D.ficus is different from that in Deinococcus radiodurain. Here, RecA, playing an important role in DNA-damage repair system, was chosen to be studies in this project. We first measure the strand exchange activity to investigate the properties of D.ficus RecA under different pH conditions, and the result shows that the highest strand exchange efficiency was found under pH 8. This optimal condition is different from E-coli RecA, and not correlated with the nuclear-protein filament stability as well. To further understand the mechanism of pH-dependent D.ficus RecA-mediated strand exchange reaction, a single molecule approach—Tethered Particle Motion (TPM) was used to elucidate the mechanism. D.ficus RecA possessing higher DNA binding affinity than E coli RecA does indicates the lower activation energy in the formation of nucleoprotein filament. However, the presence of more stable three-strand intermediates during D.ficus RecA-mediated homologous searching process impedes the completion of strand exchange. Therefore, higher strand exchange efficiency under basic condition results from the compromise between the stabilities of nucleoprotein filament and three-strand intermediate. Together, the higher average pI and the loss of partial C-terminal acidic residues evolutionally make D. ficus RecA retain activity under basic condition.

目錄
致謝 i
圖表目錄 iv
中英對照縮寫表 v
中文摘要 vii
英文摘要 viii
Chapter I Introduction 1
1-1 Deinococcus ficus (D. ficus) 1
1-2 Repair system 5
1-2-1 Base damage 6
1-2-2 Strand break 6
1-2-3 Homologous recombination 8
1-2-4 Bypass repair 8
1-3 RecA in DNA repair 9
1-4 Single molecule analysis 14
1-5 Specific aim 15
Chapter II Materials and Methods 16
2-1 D.ficus RecA ligation plasmid 的製備 16
2-2 蛋白質純化 16
2-3 Strand exchange 18
2-4 ATP hydrolysis 19
2-5 TPM DNA 製備 21
2-6 TPM 乳膠小球製備 22
2-7 TPM 反應腔室製備 23
2-8 TPM反應過程 24
2-9 分析方法 25
2-10 菌株生長測試 26
2-11 圓二色光譜 26
Chapter III Result 27
3-1 Purification of D.ficus RecA 27
3-2 活性測試 29
3-2-1 反應物偏好性 29
3-2-2 酸鹼偏好性 32
3-2-3 圓二色光譜 37
3-3 分子機制 38
3-3-1 Filament Formation Experiment 41
3-3-2 Invading Experiment 44
3-3-3 Displacement Experiment 46
3-4 生長速率測試 47
Chapter IV Discussion 49
4-1 D.ficus RecA的純化特性 49
4-2 C-terminal 缺失所影響的反應活性 49
4-3 反應活性的分子機制 50
附錄 54
Chapter V References 57


圖表目錄

Figure 1-1 系統演化族譜 2
Figure 1-2 奇異球菌屬相較於大腸桿菌的存活率 4
Figure 1-3 D.ficus 在UV照射下的反應 5
Figure 1-4 大腸桿菌之重組反應過程 7
Figure 1-5 RecA結構圖 10
Figure 1-6 RecA進行股互換反應的主要步驟 11
Figure 1-7 RecA活性於大腸桿菌及Dr的比較 13
Figure 2-1 股交換活性反應示意圖 20
Figure 2-2 ATP水解速率測量反應示意圖 21
Figure 2-3 反應腔室的製備 23
Figure 2-4 TPM 實驗設計圖 24
Figure 3-1 RecA鹽析純化 27
Figure 3-2 RecA管柱純化 28
Figure 3-3 反應物偏好性測試 30
Figure 3-4 利用螢光標定偵測股交換反應偏好 31
Figure 3-5 反應物偏好水解活性 32
Figure 3-6 酸鹼偏好股交換活性測試 33
Figure 3-7 股交換活性測試 34
Figure 3-8 水解速度測試 35
Figure 3-9 Sequence aliment 36
Figure 3-10 圓二色光譜 37
Figure 3-11 中間型產物觀察 39
Figure 3-12 股交換的主要步驟及相對應的實驗設計 40
Figure 3-13 DNA延長測試 42
Figure 3-14 酸鹼值影響纖維形成差異 43
Figure 3-15 三股中間型階段產物存在時間計算 45
Figure 3-16 股置換速率測試 46
Figure 3-17 菌株生長測試 48
表一 Buffer condition 54
表二 Primer list 55
表三 PCR condition 55
表四 exo-nuclease 反應濃度 56
表五 Km 、Vmax及Kcat 56
表六 同源搜尋中間產物穩定性 56

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