Direct Observation of RecA-Mediated Four-strand Exchange at the Single-Molecule Level

大腸桿菌在複製DNA 的過程中，因為DNA 的斷裂而造成複製過程中止，產生double-strand break，修補的途徑為同源重組反應，其中的核心步驟在於RecA 與DNA 形成的核絲結構可辨認相同序列的DNA 分子，並催化交換反應的進行。雖然細菌體的RecA 較廣泛的被研究，但真核生物體中皆已發現相似功能的蛋白存在，且交換反應的步驟大致可分為RecA 先與其中一條具有單股結構的DNA 鍵結，催化兩條相同序列的DNA 分子交換，RecA 隨後與DNA 分子脫離。同源重組反應中所牽涉到的交換反應是四股系統，以往的研究多著重於三股交換系統，且無法有效區別交換反應中各步驟的作用。論文中以單分子TPM 實驗直接觀察RecA所催化的四股交換反應，藉固定於玻片上的DNA 分子因四股交換反應發生而離去的比例，獲得四股交換反應的性質。首先，分別在ATP 及ATPγS 的條件下反應，ATPγS 是與ATP 結構相近卻無法被RecA 水解的分子，發現四股交換反應需要水解ATP。再以不同位置的單股缺口DNA 進行實驗，在我們所設計的DNA 長度下，四股交換反應沒有偏好特定位置的單股結構。並嘗試建立“n-step”動力學模型描述四股交換的反應機制，提出交換反應的進行是以相同的速率重複交換一定長度的DNA 分子。擬合結果顯示四股交換反應的速率常數與過去三股交換反應研究的速率常數值接近，當參與交換反應的DNA 分子，單股突出長度近百個鹼基對且為5''端overhang 時，四股交換反應速率大於單股突出長度較短或是3''端overhang 的其他組實驗。E. coli RecA protein is required for repairing damaged DNA through homologous recombination pathway. The assembled RecA/DNA nucleoprotein filaments catalyze the reactions that paired and exchange with homologous DNA sequence. We used single-molecule tethered particle motion (TPM) experiments to directly observe the strand exchange process catalyzed by RecA in real-time. The surface-bound DNA is a duplex DNA with a bead could be displaced due to four-strand exchange. Calculate the percentage of leaving tethers under different condition to obtain the properties of four-strand exchange. The RecA-mediated strand exchange efficiency is higher for ATP than ATPγS, suggesting that ATP hydrolysis by RecA is required to complete strand exchange. In addition, the RecA-mediate strand exchange is shown to progress either from 5’end or from 3’end in our experiment. Furthermore quantitative analysis of DNA exchanging time courses using ‘‘n-step’’ sequential mechanisms, can reveal information about the rate constant of four-strand exchange reaction and the average number of basepairs in the strand exchange cycle. The result indicates that the rate is larger for longer single-stranded gap and 5’overhang