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Title: | 利用單分子技術探討SWI5-SFR1促進DNA同源重組酶-RAD51核蛋白絲形成的機制 Single-molecule biochemical studies on the stimulation mechanism of SWI5-SFR1 complex on RAD51 presynaptic filament formation |
Authors: | CHIH-HAO LU 盧致豪 |
Advisor: | 李弘文 |
Keyword: | RAD51,SWI5-SFR1,DNA同源重組修護,單分子栓球實驗,單分子螢光共振能量轉移, RAD51,SWI5-SFR1,DNA homologous recombinational repair,smTPM,smFRET, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | 真核生物中的RAD51蛋白是驅動DNA同源重組修護反應最重要的重組酵素(recombinase);RAD51會結合到受損、裸露的單股DNA上形成核蛋白絲(nucleoprotein filament),形成RAD51-單股DNA的核蛋白絲是催化後續同源配對(homology pairing)以及股交換反應(strand exchange)的必要步驟。RAD51核蛋白絲生成反應主要可以區分為較慢的成核反應(nucleation)以及非常快速的延伸反應(extension);為了克服成核反應的高能量障壁,有許多輔助蛋白(accessory proteins)參與並加速RAD51的成核反應,以增加整體DNA同源重組修護反應的效率,其中SWI5-SFR1輔助蛋白(S5S1)被證明擁有和RAD51形成複合體(complex)、刺激RAD51所調節的DNA同源重組反應。在此篇論文中,我們利用單分子栓球實驗(single-molecule tethered particle motion, smTPM)以及單分子螢光共振能量轉移實驗(single-molecule Forster resonance energy transfer, smFRET)證明老鼠SWI5-SFR1能有效地刺激老鼠RAD51的成核反應。我們同時也證明這樣的刺激功能主要由以下幾種機制來達成:(一)減少RAD51成核單位 (nucleation unit); (二)增加RAD51對單股DNA的親和力(ssDNA affinity) ; (三)穩定RAD51所形成的核 (nuclei);然而SWI5-SFR1在另一個物種-分裂生殖酵母菌(Schizosaccharomyces pombe, Sp)-並沒有刺激成核反應的功能,但是酵母菌SWI5-SFR1擁有防止RAD51核蛋白絲解離(disassembly)的功能,且其效率比起老鼠SWI5-SFR1還要高。在不同的物種中,即使是同一種輔助蛋白,它們也會因應RAD51在物種間不同的性質和活性而有不同的調節功能:老鼠SWI5-SFR1有效地刺激老鼠RAD51的成核反應,而酵母菌SWI5-SFR1擁有防止RAD51核蛋白絲解離的功能,但最後都能達到穩定RAD51核蛋白絲的效果。 Eukaryotic RAD51 protein is essential for DNA homologous recombination in repairing doule-stranded DNA breaks. RAD51 recombinases assemble onto ssDNA to form a presynaptic filament, the required functional component for homology pairing and strand exchange reactions. This filament assembly is the committed step of homologous recombination and is subjected to regulation. Nucleation step is kinetically slow, and several accessory proteins have been identified to regulate RAD51 nucleation. SWI5-SFR1 (S5S1) is a heterodimeric accessory protein, and previous biochemical work showed that S5S1 interacts with RAD51, and stimulates RAD51-mediated homologous recombination. Our single-molecule tethered particle motion (TPM) and single-molecule fluorescence resonance energy transfer (smFRET) experiments demonstrated that mouse S5S1 interacts with mouse RAD51 to form complex, and the mRAD51-S5S1 complex efficiently stimulates the nucleation step. We also showed that mS5S1 stimulates mRAD51 nucleation by (i) reducing mRAD51 seed size, (ii) increasing mRAD51 ssDNA affinity and (iii) stabilizing mRAD51 nucleus on ssDNA. While nucleation stimulation by S5S1 is absent in fission yeast (Schizosaccharomyce pombe, Sp) system in our single-molecule work, SpS5S1 is shown to prevent SpRad51 disassembly. Different regulation strategies among species allow S5S1 to stabilize Rad51 filament efficiently. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77954 |
DOI: | 10.6342/NTU201702131 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 化學系 |
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ntu-106-R04223149-1.pdf Restricted Access | 3.56 MB | Adobe PDF |
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