利用單分子螢光共振能量轉移探討同源重組酶Dmc1核蛋白絲之構型

Characterizing Conformations of Dmc1 Recombinase Nucleoprotein Filament Using Single Molecule Fluorescence Resonance Energy Transfer

徐秉鈺(Ping-Yu Hsu)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002845

摘要

同源重組反應是DNA雙股斷裂時主要的修復途徑。在真核細胞中主要有兩個重組酶：Rad51和特定於減數分裂時作用的Dmc1。Rad51已經被證明與大腸桿菌重組酶EcRecA相似，可於單股DNA上可形成延長的核蛋白絲而Dmc1在電子顯微鏡下觀察到還具有一個特有的疊環結構。在此篇論文中，我們利用單分子螢光共振能量轉移實驗探討酵母菌體系中，ScDmc1結合於單股DNA上的構型變化。Rad51在結合至單股DNA上形成核蛋白絲時，會將在單股兩端的螢光修飾距離拉開，使訊號讀得一較低的FRET狀態；在Dmc1存在的條件下，除此之外還可以觀察到另一個使FRET較DNA本身訊號高的狀態。這個High-FRET狀態可以受到核苷酸的調控，暗示著其為一個在Dmc1催化ATP水解過程中中間體的複合構型ADP、或是不含核苷酸的Dmc1-ssDNA型態。我們進一步利用同源雙股捕捉試驗證實，無論是延長DNA的核蛋白絲、亦或是形成High-FRET的狀態，ScDmc1-ssDNA皆具有捕捉同源雙股的能力，且這兩個捕捉器對於雙股的親和力及維持雙股的穩定性相當。我們也確認這兩種Dmc1構型皆能進行後續的股交換。由於已知股交換反應需要ATP催化才可以進行，表示ADP或不含核苷酸狀態的ScDmc1-ssDNA具有黏合同源雙股的能力，但須要轉換到ATP時才能進行股交換反應。

關鍵字

同源重組 ； Dmc1 ； Rad51 ； 單分子螢光共振能量轉移

並列摘要

Homologous recombination is the major pathway to repair double-stranded break DNA damage. In eukaryotic cells, there exist two recombinases: Rad51 and meiosis-specific Dmc1. Rad51, similar to bacteria RecA recombinases, has been shown to form extended nucleoprotein filaments on ssDNA, but electron microscopy studies showed an additional stacked-ring structure for Dmc1. Here, we used single-molecule fluorescence resonance energy transfer (smFRET) experiments to study the Saccharomyces cerevisiae Dmc1-DNA complex. Rad51 binding to single-stranded DNA leads to an increase in separation of dye pairs labeled on DNA ends and results in a low FRET state. However, in the presence of Dmc1 recombinases, an additional high FRET state is observed. The high FRET state can be regulated by nucleotide cofactors, suggesting this high FRET state is a intermediate during ATP hydrolysis, ADP or no nucleotide state, promoted by Dmc1. Surprisingly, both ScDmc1-ssDNA states are biochemically functional, capable of capturing duplex DNA with similar affinity and stability with homologous duplex. Furthermore, we demonstrate that both Dmc1 states can proceed to complete strand exchange reaction, implying that potential strand annealing activity of ADP states promotes strand exchange efficiency when ATP is included.

並列關鍵字

homologous recombination ； Dmc1 ； Rad51 ； single-molecule fluorescence resonance energy transfer

參考文獻

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