臺灣博碩士論文加值系統

真核生物的 Rad51 和原核生物的 RecA 重組蛋白酵素，在DNA 利用同源性重組的修復過程中，扮演必要的角色。當DNA受損而產生雙股斷裂時，重組蛋白酵素會在DNA 上形成螺旋狀的核蛋白絲，並利用該核蛋白絲尋找同源染色體上的互補序列進行修復。單分子栓球實驗可以直接觀察 DNA長度在生化過程中的變化。我們利用單分子栓球實驗來直接觀察並了解重組蛋白酵素進行互換反應的機制。當修飾小球的 Rad51 核蛋白絲在雙股 DNA 上尋找互補序列時，一開始的布朗運動值可以被準確紀錄。並可利用 sum of two-segment 模型來模擬。本實驗即是在研究 DNA 三股交換反應中， Rad51 核蛋白絲及雙股 DNA的作用模式。我們觀察到成功地完成三股交換的 Rad51 核蛋白絲較容易以其5端位置進行初始的互換反應。我們推測 Rad51 核蛋白絲是以5端往3端的方向進行初始的交換作用，所以當一開始與雙股 DNA 碰撞的位置越靠近核蛋白絲的5端時，會有越高的機會成功完成互換。這個由 Rad51 酵素催化的結果和之前 RecA 的實驗中有相同的趨勢，在成功的三股交換反應中，發現 Rad51 及 RecA 核蛋白絲都是以其5端部分進行初始觸碰，並在三股交換反應中表現5端往3端的方向性。

Rad51 recombinases in eukaryotes and RecA recombinases in prokaryotes play an essential role in repairing damaged DNA by the homologous recombinational repair pathway. Once assembled on single-stranded (ss) DNA, Rad51 nucleoprotein filaments mediate the pairing and strand exchange with the homologous sequence. Single-molecule tethered particle motion (TPM) experiments monitor the DNA length and topology change during biochemical processes, and allow us to study the mechanistic details of DNA recombination processes. In the Rad51 invading strand experiments, beads were labeled on the invading ssDNA with sequence homologous to the surface-anchored duplex DNA. When bead-labeled Rad51 nucleoprotein filaments first interacted with the surface-anchored DNA, the initial Brownian motion (BM) amplitude which was resulted from the combined length contribution of the Rad51 nucleoprotein filament and surface anchored duplex DNA can be detected. The initial Brownian motion provides information on the initial contact point of the Rad51 nucleoprotein filament as well as its polarity preference for the stable synapses formation. A sum of two-segment model successfully describes the distribution of initial BM values. For transient events, the synaptic complex formation initiated at random position with no end preference. For events that drive successfully into the final strand exchange product, 5′-end segment of Rad51 nucleoprotein filament was preferentially used. Our studies suggest that Rad51 nucleoprotein filaments carry out initial strand exchange in the 5′-to-3′ direction.

CHPTER 1 : INTRODUCTION 1
1-1 Homologous recombination and Rad51 recombinases 1
1-2 Strand exchange direction mediated by Rad51 and RecA 3
1-3 Tethered particle motion in single molecule experiments 7
1-4 The invading strand experiment in tethered particle motion 11
CHPTER 2 : MATERIAL AND METHOD 13
2-1 Protein expression and purification 13
2-2 DNA substrates 13
2-3 Single molecule tethered particle motion measurement and data analysis 22
2-4 The invading strand experiment 23
2-5 Sum of two-segment model 26
CHPTER 3 : RESULT .30
3-1 The initial Brownian motion of Rad51 nucleoprotein filaments 30
3-2 The Rad51 nucleoprotein filaments with no ATP hydrolysis 34
3-3 The three-strand exchange direction of Rad51 37
CHPTER 4 : DISCUSSION AND CONCLUSION 40
4-1 The initial Brownian motion of strand exchange mediated by RecA 40
4-2 The strand exchange direction mediated by Rad51 43
4-3 The 5′-to-3′ strand exchange direction and the proposed model 46
APPENDIX 48
REFERENCE 51

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