以單分子螢光技術調查RecBCD解螺旋酶兩個不同ATP水解酶的相關性

Abstract

RecBCD為啟動大腸桿菌同源重組反應的三聚體酵素， RecBCD同時具有單股DNA的水解活性以及兩個不同方向性的DNA解螺旋酶活性。RecBCD分別由RecB(3'-to-5' DNA解螺旋酶、核酸酶)、RecC(辨認chi序列：5'-GCTCCTCC-3')以及RecD(5'-to-3' DNA解螺旋酶)。先前利用單分子栓球實驗證實RecBCD中的RecB以及RecD不僅是雙股DNA解螺旋酶，也可以分別在單股DNA上移位。先前研究在RecBCD三聚體形成時，RecBCD 3到5及5到3的移位速率皆產生明顯的變化，指出RecBCD形成三聚體時，RecB與RecD之間可能存在著兩個次單元之間的交互作用以及協同性。由於解螺旋酶作用是利用ATP水解來提供DNA解螺旋的能量，所以大部分解螺旋酶也同時具有ATP水解酶的能力。這裡我們利用全反射螢光顯微技術來偵測RecBCD在DNA上解旋時，RecB以及RecD兩個解螺旋酶之間的協同性。本篇論文即是利用量測RecB及RecD兩個ATP水解酶的活性相關性來研究RecB及RecD解螺旋酶的交互作用。我們利用量測單一個螢光ATP結合於RecBCD的ATP水解酶的時間長短來測量其ATP水解速度。由野生型RecBCD以及RecBCD* K177Q的實驗結果推測RecB以及RecD的ATP結合時間是不同的，但結果顯示在野生型RecBCD水解DNA時，只能觀測到單一個RecBCD特定的ATP水解速度，而且螢光ATP的結合時間與ATP的濃度呈正相關，代表RecB與RecD同時作用時的相關性使得兩者具有相同的ATP結合時間。

RecBCD is a trimeric enzyme complex that initiates homologous recombinational repair in E. coli. RecBCD complex contains single-stranded DNA nuclease and two DNA helicases. In these two helicases, RecB is a 3'-to-5' single-stranded DNA translocase and RecD is a 5'-to-3' one. Formation of RecBCD trimeric complex was shown to stimulate both 3'-to-5' and 5'-to-3' translocation activities, implying interaction and coordination of RecB and RecD helicases in the trimeric complex. ATP hydrolysis is coupled to the unwinding and translocation process during the catalytic step of DNA helicases, so most DNA helicases are ATPases. Here, we used total internal reflection fluorescence microscopy to directly observe and analyze individual Cy3-labeled ATP binding and turnover events during RecBCD unwinding at the single-molecule level. This allows us to characterize ATPase activities of both RecB and RecD as well as their coordination.Comparison of Cy3-ATP bound time between RecBCD and RecBCD* K177Q shows different ATP bound time.Though we can only measure one ATP bound time of wild type RecBCD, we found positive correlation of ATP concentration and ATP bound time. It implies the coordination between RecB and RecD synchronizes both ATPase catalytic rates.