臺灣博碩士論文加值系統

同源重組酶Dmc1及Rad51在DNA雙股斷裂的修復中扮演重要角色。在同源重組修復過程中，重組酶會結合到受損DNA裸露出的3′單股DNA，形成核蛋白絲 (pre-synaptic filament)，並尋找同源的雙股DNA，以同源雙股DNA作為修復模板，進行股交換反應 (strand exchange)，完成高準確度的DNA修復。大部分真核生物同時具有Rad51及Dmc1兩個同源重組酶，兩者無論是序列、結構及功能，都具有高度相似性，其中Rad51會在細胞分裂及減數分裂細胞時被表達，但是Dmc1只出現於減數分裂的細胞中。關於減數分裂時同時需要這兩個同源重組酶的參與，雖然已有不少遺傳學或生化實驗提供線索，但至今仍然沒有明確的機制。在此篇論文中，我們利用單分子栓球實驗 (single-molecule tethered particle motion) 比較酵母菌 (Saccharomyces cerevisiae) Rad51及Dmc1形成核蛋白絲的動力學性質。核蛋白絲生成的過程中，在單股DNA上形成核種 (nucleation) 為速率決定步驟。我們發現到ScRad51與ScDmc1的成核動力學有明顯的差異: (1) ScRad51和ScDmc1成核時，對DNA結構具有不同的結合偏好；(2) ScRad51核蛋白絲形成的成核速率比ScDmc1來的快。ScRad51對於單股DNA的結合親和力比ScDmc1優越，因此ScRad51傾向成核於單股DNA。然而我們意外觀察到ScDmc1對於5′ 單/雙股DNA交界 (duplex DNA/ss DNA junctions) 具有顯著的偏好性，推測ScDmc1傾向成核於這些位置，並由5′ 端單股DNA往3′ 端延伸核蛋白絲 (extension)。這個5′ 單/雙股DNA交界的偏好性也存在小鼠的DMC1，顯示這個DNA結構偏好的一般性及重要性。另外，加入部分ScRad51核蛋白絲的DNA能有效提升ScDmc1成核速率，反應出Dmc1在有效成核過程中需要結構上的對接點 (docking sites)，如單/雙股DNA交界或其他蛋白。我們的研究指出Rad51很可能利用其高DNA親和力，快速形成短的核蛋白絲，來幫助Dmc1在減數分裂中有效形成核及組裝蛋白絲。

Dmc1 and Rad51 recombinases play important roles in the DNA double strand break repair. During the homologous recombination, recombinase binds to the resected damaged DNA to form a nucleoprotein filament, responsible for homology pairing and strand exchange. Rad51 and Dmc1 both exist in most eukaryotic cells, sharing similar amino acid sequences, structures and functions. However, Rad51 is expressed in both mitotic and meiotic cells, but Dmc1 is a meiosis-specific recombinase. The underlying mechanism of this differential requirement is unclear. Here, we utilized single-molecule tethered particle motion experiments to compare the kinetics of nucleoprotein filament assembly of Saccharomyces cerevisiae Rad51 and Dmc1. Nucleation on single-stranded DNA (ssDNA) is the rate-limiting step of the nucleoprotein filament assembly. We found distinct differences of these two recombinases: (1) ScRad51 and ScDmc1 have different nucleation preferences of DNA structures. (2) The nucleation rate of ScRad51 is much faster than ScDmc1, indicating that ScRad51 has better ssDNA binding affinity, and preferentially assembles on ssDNA. However, ScDmc1 preferentially nucleates on DNA substrates with duplex DNA/ssDNA junction containing a 3′-ssDNA overhang, as it allows filament extension from 5′-to-3′ direction. Same 5′ ds/ssDNA junction preference is also found in mouse DMC1, suggesting the general and important role of this nucleation site preference for the Dmc1 recombinase. Surprisingly, in the DNA substrates containing short discontinuous patches of ScRad51, ScDmc1 assembly is stimulated. Our data imply that the nucleation of ScDmc1 on ssDNA requires docking sites, such as duplex DNA/ssDNA junction or Rad51 binding on ssDNA. Higher ssDNA affinity of ScRad51 might offer the nucleation docking sites for ScDmc1 assembly during meiosis.

謝辭 i
摘要 iii
ABSTRACT iv
目錄 v
圖目錄 vii
表目錄 viii
第1章 緒論 1
1-1 減數分裂中同源重組機制 1
1-2 研究動機 7
第2章 實驗設計與方法 9
2-1 蛋白質純化與保存 9
2-2 DNA製備 9
351/dT90, 351/dT135 9
351/dT(45)n (n=2、3) DNA 及351/dT(45)2-flap DNA 10
351/dT(37+29+36) 10
2-3 緩衝溶液配方 13
2-4 單分子栓球實驗 13
2-5 反應槽製備 14
一般反應槽製備 14
矽烷化反應槽製備 14
2-6 顯微鏡參數 15
2-7 數據分析 15
布朗運動分析 15
成核速率分析 16
第3章 實驗結果與討論 19
3-1 比較ScRad51及ScDmc1 與DNA之結合 19
3-2 hDMC1及mDMC1也具有聚縮及回升現象 22
3-3 ScDmc1之延伸與回升具有類似動力學性質 24
3-4 ScRad51與ScDmc1具有不同成核偏好性 26
3-5 ScDmc1傾向成核於5''單/雙股DNA交界處 30
3-6 ScDmc1於5''單/雙股DNA交界處的成核偏好性不受離子濃度影響 33
3-7 mRAD51與mDMC1也具有不同成核偏好性 35
3-8 ScRad51作為ScDmc1核蛋白絲形成的輔助蛋白 39
第4章 結論 45
4-1 結果討論 45
比較Rad51及Dmc1 與DNA之結合 45
Dmc1與Rad51具有不同成核偏好性 46
ScRad51作為ScDmc1核蛋白絲形成的輔助蛋白 48
4-2 未來展望 52
參考文獻 53

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