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Title: | 直接觀察RecA重組酶形成核蛋白絲時雙股去氧核醣核酸螺旋結構的旋轉過程 Direct observation of DNA rotation during RecA nucleoprotein filament formation |
Authors: | Yu-Tsung Lee 李宥宗 |
Advisor: | 李弘文 |
Keyword: | RecA重組酶,核蛋白絲,金奈米棒,暗場顯微鏡, RecA,nucleoprotein filament,gold nanorod,darkfield microscopy, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | 在生物體內,同源重組反應 (homologous recombination) 是一個關鍵的生化反應,當DNA在複製過程中發生斷裂時進行修補,以維持基因的完整性。在真核生物體進行減數分裂時,同源重組反應的發生,主要是維持基因的異質性。原核生物中的RecA重組酵素和真核生物內有相似功能的蛋白,會和DNA形成核絲蛋白結構,並且尋找互補序列的DNA分子催化和起始同源重組反應。但是RecA和DNA交互作用形成核絲蛋白的機制尚未被完全了解。因此,我們設計了幾個單分子實驗去觀察當RecA酵素在DNA上形成核蛋白絲時,所造成雙股DNA螺旋結構部份解旋的現象。在目前的文獻中,研究團隊大都在DNA的末端接上標誌以放大解旋的過程。本論文主要透過金奈米棒接上DNA的末端,利用暗場顯微技術照射,並且在收光位置裝設在空間上互相正交的二片偏振片,觀察金奈米棒在x軸和y軸上的投影強度變化。藉由投影在二軸上的強度比例,可以獲得金奈米棒在空間上和x軸的夾角。此外,我也在DNA的末端接上一組雙球標誌,此組雙球標誌是由直徑為1微米的磁球和直徑為40奈米的量子點所組成,因磁球散射光和量子點放光在波長上的差異,利用dualview系統分開此二種放光,在攝影機上同時紀錄磁球和量子點的軌跡。此外,當RecA蛋白在雙股DNA上形成核蛋白絲時,DNA會部份解開雙股螺旋結構。架設磁鑷子抑制雙球標誌在溶液中的布朗運動,降低紀錄量子點軌跡時,來自於布朗運動的干擾。同時得到此組雙球的軌跡,進而得到形成核蛋白絲時所造成的螺旋結構解旋資訊。 Homologous recombination is a key biochemical process, in which repairing damaged DNA and subsequently maintaining genome integrity takes place among organisms. The recombinase, RecA, in prokaryote plays an important role in initiating and catalyzing the recombination reaction during DNA replication and repairing. It is of great interest to unveil the interaction between RecA and DNA as RecA forming nucleoprotein filament on DNA. To understand how this proceeds, we developed a few single-molecule experiments to detect the rotation of the nucleoprotein filament during the RecA assembly process. In general, this rotation process can be visualized by attaching an object to amplify its rotation process dictated by helical property of the DNA. Here, we demonstrated a single gold nanorod (GNR) method, along with dark field illumination microscopy. By analyzing alternative variations of polarization intensity in x and y directions scattering from gold nanorod coupled to DNA, we can hence resolve angular information during RecA polymerization in real time. We also developed a bead dimer experiment in which one of the end of a single DNA molecule was attached a dimer bead complex composed of a magnetic fluorescent bead and quantum dots with a broad absorption band and narrower fluorescence emission. This method allows us to track the motion of two beads simultaneously during the RecA assembly process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22976 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 化學系 |
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ntu-100-1.pdf Restricted Access | 1.88 MB | Adobe PDF |
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