利用單分子螢光方法觀察RAD51及其同源蛋白之交互作用

Abstract

RAD51同源同種物（paralog）是參與同源重組（homologous recombination）修復的重要蛋白。BCDX2是由RAD51同源同種物RAD51B、RAD51C、RAD51D、及XRCC2組成的蛋白複合體，被認為能協助RAD51聚集。我使用了三種單分子螢光方法了解BCDX2是否影響RAD51和DNA的結合。螢光標記蛋白和零模波導（zero-mode waveguides）的螢光實驗發現BCDX2和DNA的結合反應是一動態過程，且在RAD51存在時結合能力下降。單分子螢光共振能量轉移（FRET）實驗發現，在含有BCDX2的情況下，RAD51蛋白絲對單股和雙股DNA所產生的拉伸效果並未受到影響。然而，BCDX2在有ATP的條件之下提昇了RAD51在雙股DNA上的蛋白誘導之螢光增強效應（protein-induced fluorescence enhancement）。此效應與反應時間相關，且可能反映出ATP水解反應的參與。這些觀察為未來研究BCDX2在同源重組中扮演的角色，提供新的資訊。

RAD51 paralogs are important proteins that participate in homologous recombination repair. One of the heteromeric complexes BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) is hypothesized to stimulate RAD51 foci formation. I used three single-molecule fluorescence tools to investigate how BCDX2 affects the RAD51 binding. Using fluorescently labeled protein and zero-mode waveguides, BCDX2 binding to DNA was found to be dynamic and reduced by RAD51. Single-molecule fluorescence resonance energy transfer (FRET) showed that BCDX2 does not alter the DNA extension property of RAD51 on single-strand DNA (ssDNA) or double-strand DNA (dsDNA). However, protein-induced fluorescence enhancement (PIFE) results showed that BCDX2 increased the PIFE effect of RAD51 on dsDNA but not ssDNA in the presence of ATP, in a time-dependent manner. This PIFE increase is potentially linked to ATP hydrolysis. These observations provide new information to elucidate the roles of BCDX2 in homologous recombination.