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  • 學位論文

單分子技術觀察DNA錯誤配對辨認蛋白 MutS 如何抑制 RecA 重組酶進行部分同源股交換反應

Investigate How Mismatch Repair Protein MutS Inhibits RecA-Catalyzed Homeologous Strand-Exchange Reaction Using a Single-Molecule Method

指導教授 : 李弘文

摘要


在DNA複製的過程中,可能會受到一些破壞而需要修復機制,而同源重組反應即是其中一種重要的修復機制。在大腸桿菌等原核生物中,其中核心的步驟在於RecA蛋白與DNA形成的核絲結構可辨認相同序列的DNA分子,並透過股交換反應來修復DNA。先前的研究指出,即使在欲交換的DNA序列上面有與原本單股DNA有不配對的序列,仍可進行部分同源股交換反應,生成含有錯誤配對的DNA產物。而錯誤辨認蛋白MutS可以抑制這樣的部分同源股交換反應,但其中抑制的原理以及機制卻仍不明。除了傳統的電泳生化分析技術,我們也使用了單分子栓球實驗去觀察部分同源股交換反應,以及MutS蛋白在其中的角色。首先,我們發現在進行RecA催化部分同源股交換反應當中,即使在接近的錯誤配對比例之下,錯誤配對的連續性是影響RecA催化部分同源股交換反應的重要因素。第二,我們使用線性且較短的DNA基質組進行MutS蛋白抑制部分同源股交換反應,除了反應被抑制以外,也看見更高分子量的副產物出現,我們推測此副產物可能是股交換反應的中間產物,三股DNA的結構。接著,我們也以單分子栓球實驗去測量RecA核蛋白絲與雙股DNA的作用,結果顯示在我們的解析度之下,MutS蛋白在這一步驟並無影響,這一系列的實驗也提示了我們三股DNA是MutS蛋白抑制部分同源股交換反應的研究方向。

並列摘要


Homologous recombination (HR) plays an important role in meiosis and DNA damage repair. In prokaryotes, RecA nucleoprotein filaments search complementary DNA sequence, and carry out the strand-exchange reaction of essential steps in HR. Moreover, RecA can catalyze strand-exchange reaction even if DNA sequence not totally homologous, i.e. homeologous. Previous studies indicate that mismatch repair protein, MutS, inhibits RecA-mediated homeologous recombination. However, the inhibition mechanism remains unknown. We found the DNA mismatch density plays an important role in inhibiting the homeologous strand-exchange reaction. Using gel-shift electrophoresis assay, an intermediate state of strand exchange reaction with higher molecular weight appeared in the presence of MutS. This suggests that MutS proteins promote the accumulation of the reaction intermediate, thus reduce final reaction products. Using single-molecule tethered particle motion experiments, we measured interaction between RecA filaments and homeologous DNA substrates in the presence of MutS. These experiments offer information about how MutS inhibition might take place.

並列關鍵字

RecA TPM Single molecule DNA repair MutS Homeologous recombination

參考文獻


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