以單分子方法探討人類Dmc1蛋白在進行DNA股交換時的核蛋白結構

Investigating Human Dmc1 Nucleoprotein Structure During DNA Strand Exchange Using Single Molecule Methods

呂易翰

同源重組 ； DNA 修復 ； Dmc1 ； 單分子栓球實驗 ； 自相關函數 ； homologous recombination ； DNA repair ； Dmc1 ； tethered particle motion ； autocorrelation

臺灣大學化學研究所學位論文

2013年

碩士

李弘文

繁體中文

當 DNA 因為雙股斷裂 (double-strand break, DSB) 而受損時，細胞會進行一個稱為同源重組(homologous recombination, HR) 的反應來對受損的 DNA 進行修復。同源重組反應是由同源重組酶催化進行的。當此反應進行時，同源重組酶須要先和 ATP 結合，接著聚集在單股 DNA (single-stranded DNA, ssDNA) 上形成核蛋白絲結構。股交換反應完成後，同源重組酶會水解 ATP 接著從 DNA 上脫離。Dmc1在真核細胞內是專門負責進行減數分裂時期同源重組的蛋白質。和其他兩個會形成延長的核蛋白絲的同源蛋白質 RecA 及Rad51 不同的是，Dmc1 在利用電子顯微鏡的研究中被觀察到有兩種不同的結構，其一是延長的核蛋白絲 (extended nucleoprotein filament)，另一種則是以堆疊環狀 (stacked ring) 的型式和 DNA 結合。在本研究中，我們使用單分子拴球技術 (tethered particle motion, TPM) 來觀察Dmc1 在催化股交換反應進行時的即時狀態，並發現 Dmc1 於此過程中並不會造成延長的三股 DNA 中間產物結構產生。即使我們加入可以藉由抑制 ATP 水解而穩定核蛋白絲結構的鈣離子也不會影響此結果。 我們更進一步的利用自相關函數來分析反應時的時間對布朗運動趨勢圖來證實了 DNA 和 Dmc1 的交互作用。

Cells utilize homologous recombination (HR) to repair the DNA double-strand break (DSB). The reaction is conducted by a kinds of protein called recombinase. While conducting the HR reaction, recombinase need to bind with ATP and then assemble on single-stranded (ss) DNA to form nucleoprotein complex. After the strand exchange completed, ATP was hydrolyzed and recombinase disassemble from DNA. Dmc1 is a meiosis-specific recombinase responsible for HR in eukaryotic cell. Unlike other two Dmc1 homolog RecA and Rad51, which have been proved to form an extended nucleoprotein filament, Dmc1 was showed to have two kinds of structure of nucleoprotein filament by electron microscopy studies, one is extended nucleoprotein filament and another is stacked ring structure. In this study we use tethered particle motion (TPM) to observe the real time behavior while Dmc1-catalyzed strand exchange reaction was processing and found that Dmc1 does not form an extended three strand intermediate, even though we add more calcium to inhibit ATP hydeolysis and stabilize the Dmc1 filament. So we further utilize the autocorrelation function to analyze the reaction timetrace and prove the DNA-protein interaction.

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