RecA同源重組酶調控DNA-RNA複合體的機制研究

Abstract

大腸桿菌的同源重組酶RecA在DNA雙股斷裂的修復過程中扮演重要角色。在同源重組的修復過程中，RecA蛋白需先與單股DNA結合形成核蛋白絲，隨後尋找完整且同源互補的雙股DNA進行股交換反應。在這過程中，RecA蛋白與入侵股、離去股和互補股皆有不同程度的交互作用。近期的研究裡發現，同源重組蛋白可以調控DNA-RNA複合體的生成與代謝過程，但RecA與RNA的交互作用及其機制尚未被釐清。這篇論文利用生化分析方法與兩種單分子技術研究RecA蛋白與單股RNA (RecA-ssRNA) 的交互作用，發現RecA與ssRNA親和性與穩定性比RecA與ssDNA低，以致於RNA作為入侵股進行股交換的低反應效率。我們也利用螢光共振能量轉移實驗觀察RNA做為互補股與離去股在動力學上的差異，發現RecA蛋白分別與RNA股的低親和性，直接影響股交換反應的速率。在利用單分子栓球分析方法探討RNA作為離去股之入侵反應中，發現同源重組蛋白仍具有促使股交換反應進行之能力，並且與典型反應同樣擁有5端的入侵偏好性以及相似的中間體滯留時間，指出此二反應具有相似的反應機制。由於DNA-RNA複合體的存在反應到基因體的不穩定，RecA蛋白可以催化ssDNA取代雙股中RNA鏈的反應，顯現出RecA同源重組酶具有調控DNA-RNA複合體代謝之功能。

RecA recombinase plays an essential role in double-strand DNA break repair (DSBR) in E. coli. RecA participates in every step of homologous recombination, including presynapsis, synapsis, and postsynapsis. At the first step, RecA binds to single-stranded DNA to form nucleoprotein filament responsible for searching homologous double-stranded DNA, and for carrying out the following strand exchange reaction. During the synaptic stage, RecA protein interacts with both the invading ssDNA as well as dsDNA (leaving strand and complementary strand). It has been recently suggested that recombinases also involve in regulating the formation and stability of DNA-RNA hybrids, but the mechanism has not been elucidated. We used both ensemble-based and single-molecule methods to study reactions involving RecA protein and RNA. We showed that RecA-ssRNA has lower affinity and stability, directly resulting in the inefficient recombination reaction when RNA served as an invading strand. Using two ensemble-based fluorescence resonance energy transfer experiments, we observed the kinetics associated with the pairing and strand displacement steps. We showed that the lower affinity of RecA-RNA affects the strand exchange progresstion if RNA strand is the complementary strand. When RNA is the displaced strand, the homologous recombination can proceed and has the same 5'-end invasion preference seen in the typical DNA substrates. Together, we proposed that RecA can regulate the metabolism of DNA-RNA hybrid.