利用單分子螢光共振能量轉移研究RecA E38K核蛋白絲之動力學

RecA蛋白在雙股DNA斷裂所引發的同源重組修復中扮演相當關鍵的角色，在同源重組過程中，RecA核蛋白絲的形成被認為是同源重組過程中的重要調控步驟，而形成RecA核蛋白絲主要包含緩慢的成核作用以及快速的延伸作用。核蛋白絲會在其他輔助蛋白的協助下，尋找未受損的雙股DNA之互補序列，並進行股交換反應以修復受損的DNA。研究發現在高劑量紫外光照射下存活率較高的RecA蛋白突變型E38K，比起RecA蛋白能在更短的時間內與單股DNA進行成核反應。在本論文中，利用單分子螢光共振能量轉移的實驗以及隱馬可夫模型，研究RecA蛋白和RecA蛋白突變型E38K在不同長度的單股DNA上之成核作用，而實驗結果發現比起RecA蛋白，RecA蛋白突變型E38K在相同長度的單股DNA上有較高的成核頻率，且可以透過更小的成核單位與單股DNA結合。此外，實驗結果也顯示RecA蛋白突變型E38K可能以單體為單位，從穩定的核蛋白絲尾端脫離或結合。

關鍵字

RecA ； RecA蛋白突變型E38K ；同源重組；單分子螢光共振能量轉移

並列摘要

RecA plays a key role in homologous recombination pathway to repair double-stranded DNA break damage. In homologous recombination, the assembly of RecA onto the ssDNA to form nucleoprotein filaments includes a slow nucleation and a rapid extension step. The assembly of RecA-ssDNA nucleoprotein filaments is the key regulatory step for homologous recombination, and the assembled nucleoprotein filament is responsible for mediating DNA homology search and downstream strand exchange. The previous study showed that a recA strain surviving high doses of UV radiation includes a dominative E38K point mutation. Single-molecule biochemical studies showed that E38K mutants have faster kinetics in forming nucleoprotein filaments compared to wild-type RecA. In this study, we used a single-molecule fluorescence resonance energy transfer (smFRET) experiment and hidden Markov analysis to study the assembly of wild-type RecA and RecA E38K at different ssDNA length (for wild-type RecA, (dT)n, n = 19, 21, 23, 27; for RecA E38K, (dT)n, n = 9, 16-23). We found that RecA E38K has smaller nucleation unit and higher nucleation frequency at the same ssDNA length ((dT)n, n = 19, 21, 23) compared to wild-type RecA. We also showed that RecA E38K filament likely assemble and disassemble with one monomer at the 5′ end of nucleoprotein filament.

並列關鍵字

RecA ； RecA E38K (recA730) ； homologous recombination ； smFRET

參考文獻

1 Story, R. M., Weber, I. T. & Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318-325 (1992).

2 Story, R. M. & Steitz, T. A. Structure of the recA protein-ADP complex. Nature 355, 374-376 (1992).

3 Cox, M. M. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol 8, 127-138 (2007).

4 Di Capua, E., Engel, A., Stasiak, A. & Koller, T. Characterization of complexes between recA protein and duplex DNA by electron microscopy. J Mol Biol 157, 87-103 (1982).

5 Egelman, E. H. & Yu, X. The location of DNA in RecA-DNA helical filaments. Science 245, 404-407 (1989).

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利用單分子螢光共振能量轉移研究RecA E38K核蛋白絲之動力學

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