RecA核蛋白絲於SSB-ssDNA上的形成過程包含RecA-SSB交互作用與SSB構型轉換

Abstract

RecA同源重組酶在DNA同源重組修復機制中負責催化同源配對和股交換反應。DNA修復的第一步，RecA必須和單股DNA結合蛋白 (SSB) 競爭single-stranded DNA (ssDNA) 以形成RecA核蛋白絲。過去的研究認為，RecA蛋白在這個過程中會採取較為被動的策略：等待直到一段ssDNA從SSB上裸露出來，接著在這段ssDNA上成核，然後延伸形成完整的核蛋白絲。在這個研究中，我們使用單分子拴球實驗 (TPM) 實時觀察單一RecA核蛋白絲在SSB-ssDNA上形成的過程。RecA核蛋白絲形成時會將ssDNA拉直，反應在拴球布朗運動的增加上。藉由改變DNA中ssDNA的長度 (60、65、70、72、75、78、80、90、100 nt) ，同時控制SSB的數量，使ssDNA上只會有一個65-nt mode SSB，我們發現RecA E38K的成核時間並沒有與ssDNA長度呈現顯著的負相關性。相反地，其成核時間隨著ssDNA上的SSB數目不同而改變 (70、135、200、264 nt，對應到1、2、3、4個SSB) 。當在反應中加入free SSB時，我們發現RecA E38K需要更長的成核時間和延伸時間，反應出來的拴球布朗運動改變量也比較小，意味著SSB構型轉換可能參與在RecA E38K的成核過程，同時影響成核速度、延伸速度與核蛋白絲長度。我們的實驗結果揭示了一個意想不到的RecA核蛋白絲形成機制：主動成核，其中包含了直接的RecA-SSB-ssDNA交互作用和SSB構型轉換。

RecA recombinase catalyzes the homology pairing and strand exchange reactions in homologous recombinational repair. RecA must compete with single-stranded DNA binding proteins (SSB) for single-stranded DNA (ssDNA) substrates to form RecA nucleoprotein filaments as the first step of the repair process. Previously, it has been suggested that RecA competes with SSB by binding and extending onto the free ssDNA region. In this study, we monitored individual RecA filament formation on SSB-ssDNA by tethered particle motion (TPM) technique in real-time. Binding of RecA on the SSB wrapped-ssDNA extends the DNA substrate, visible by the increase in bead Brownian motion in TPM imaging. By varying the length of ssDNA gap from 60 to 100 nucleotides (60, 65, 70, 72, 75, 78, 80, 90, 100), in which only one SSB binding is allowed using the 65mer wrapping mode, we found the nucleation time of RecA E38K to form extended nucleoprotein filament lacks apparent ssDNA gap size dependence. Instead, the nucleation time changes when DNA substrates with 1, 2, 3 and 4 SSB bound (70, 135, 200, 264 nucleotides) were used. Longer nucleation time and shorter RecA filament length were found when free SSB was present, indicating the involvement of SSB binding mode conversion. Our data have revealed an unexpected RecA filament formation mechanism on SSB-ssDNA in which direct RecA-SSB-ssDNA interaction and SSB binding mode conversion are involved.