利用單分子螢光方法觀察RAD51及其同源蛋白之交互作用

Using Single-Molecule Fluorescence Methods to Study the Interaction of RAD51 and Its Paralogs

李子于(Tzu-Yu Lee)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202203886

摘要

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。

關鍵字

同源重組 ； RAD51 ； RAD51同源蛋白 ； BCDX2蛋白複合體 ； 單分子螢光

並列摘要

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.

並列關鍵字

homologous recombination ； RAD51 ； RAD51 paralogs ； BCDX2 complex ； single-molecule fluorescence

參考文獻

Filippo, J. S., Sung, P. Klein, H. Mechanism of Eukaryotic Homologous Recombination. Annu. Rev. Biochem. 77, 229–257 (June 2008).

Symington, L. S. Gautier, J. Double-Strand Break End Resection and Repair Pathway Choice. Annu. Rev. Genet. 45, 247–271 (Dec. 2011).

Lisby, M., Barlow, J. H., Burgess, R. C. Rothstein, R. Choreography of the DNA Damage Response. Cell 118, 699–713 (Sept. 2004).

Wang, X. Haber, J. E. Role of Saccharomyces Single-Stranded DNA-Binding Protein RPA in the Strand Invasion Step of Double-Strand Break Repair. PLoS Biol. 2, e21 (Jan. 2004).

Sung, P., Krejci, L., Komen, S. V. Sehorn, M. G. Rad51 Recombinase and Recombination Mediators. J. Biol. Chem. 278, 42729–42732 (Oct. 2003).