以單分子光鉗系統探討SWI5-SFR1調控老鼠RAD51核蛋白絲形成動力學之研究

RAD51 催化的同源重組修復反應是真核生物體內很重要的 DNA 修補機制，在含有 ATP 的情況下， RAD51 可與單股 DNA 結合並形成核蛋白絲，此核蛋白絲會啟動同源重組修復反應，以修復致命的 DNA 雙股斷裂。 SWI5-SFR1 複合體已被報導具有穩定 RAD51 核蛋白絲的功能，但其穩定核蛋白絲的機制尚未明瞭。本研究強化並提高現有的單分子光鉗技術的穩定度，包括引入 PID 控制器進行雷射強度控制、使用能量譜密度分析進行雜訊偵測，並在實驗過程中同步進行定力鉗實驗操作與影像擷取，以利事後影像處理扣除雜訊，成功達到 1 nm 解析度。並利用此技術試圖研究老鼠種 RAD51 形成核蛋白絲的過程，包括與 DNA 結合以及自 DNA 上脫離的動力學性質，如何受到 SWI5-SFR1 複合體的調控。實驗發現在含有 ATP 的情況下， SWI5-SFR1 可提高 mRAD51 與雙股 DNA 結合的速率 kext，但並不影響 mRAD51 自雙股 DNA 上脫離的速率 kdis。另一方面，SWI5-SFR1 可提高 mRAD51 與單股 DNA 結合的速率 kext，同時抑制 mRAD51 自單股 DNA 上脫離的反應 kdis。此調控功能暗示 SWI5-SFR1 不只可以有效的穩定 mRAD51 核蛋白絲，亦有可能使 mRAD51 在股交換反應完成後，迅速地脫離 DNA，使其能夠進行下一步的修復反應。

關鍵字

同源重組； DNA 修復； RAD51 ； SWI5-SFR1 ；單分子生物物理；光鉗

並列摘要

Homologous recombination catalyzed by RAD51 recombinases is a crucial DNA repair pathway in eukaryotes. In the presence of ATP, RAD51 assembles on single-stranded DNA to form nucleoprotein filaments, and initiates homologous recombinational repair of DNA double-stranded breaks. The SWI5-SFR1 complex has been found to regulate RAD51 filament assembly and enhance strand exchange activity, but the detailed mechanism is not clear. Here we improved our home-built optical tweezers platform to 1 nm resolution, and utilized it to study the assembly and disassembly dynamics of mRAD51 filaments in the presence of SWI5-SFR1 complex. In the case of double-stranded DNA, mRAD51 assembly process is stimulated in the prescence of SWI5-SFR1, but the disassembly process is not affected. On the other hand, mRAD51 assembles onto single-stranded DNA with an enhanced rate in the prescence of SWI5-SFR1, and the disassembly process from ssDNA is suppressed by the SWI5-SFR1 complex. This indicates that the SWI5-SFR1 stabilization function happens in the mRAD51 nucleoprotein filament formation onto single-stranded DNA, while SWI5-SFR1 only alters the kext of double-stranded DNA filament formation, not kdis. These regulatory functions of SWI5-SFR1 imply not only efficient stabilization of mRAD51 nucleoprotein filament during strand exchange, but also offer efficient mRAD51 turnover once the reaction is completed.

並列關鍵字

homologous recombination ； DNA repair ； RAD51 ； SWI5-SFR1 ； single molecule biophysics ； optical tweezers

參考文獻

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國際替代計量

以單分子光鉗系統探討SWI5-SFR1調控老鼠RAD51核蛋白絲形成動力學之研究

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