探究RecA酵素在SSB蛋白存在下與單股DNA競爭機制

Investigating RecA Filament Assembly in the Presence of SSB at Single-Molecule Level

曾子綾(Tzu-Ling Tseng)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02563

摘要

大腸桿菌中的RecA酵素在DNA受到外界刺激而產生雙股螺旋斷裂時所進行的同源重組修復過程扮演極重要的角色。在同源重組的修復過程中，首先RecA酵素必須先與單股DNA做結合形成核蛋白絲，接著尋找完好且能夠同源互補修飾的雙股DNA以進行股交換反應。在DNA的代謝過程會產生極不穩定的單股DNA，須利用單股DNA結合蛋白 (SSB) 穩定與保護單股DNA的結構，所以當RecA酵素需要在單股DNA上形成核蛋白絲的過程時，必須先想辦法將單股DNA上的SSB取代下來。實驗目的想要探討SSB存在於單股DNA上會造成阻礙使RecA酵素無法有效成核在單股DNA上抑或是RecA酵素能夠主動的取代纏繞著單股DNA的SSB以形成核蛋白絲。
SSB能夠穩定且保護單股DNA，但我們對於RecA酵素如何取代SSB而與單股DNA形成核蛋白絲的反應機制過程是模糊的。為了比較大腸桿菌中RecA酵素在SSB存在下與單股DNA結合的作用差異，我們利用RecA酵素突變體E38K來做比較，突變體E38K在與單股DNA做結合時不需要中介物RecFOR的幫助便能夠輕易的取代SSB並在正確位置與單股DNA結合。我們利用單分子拴球實驗的架設直接觀測RecA酵素與個別的DNA做結合時布朗運動值的改變，所使用的基質為無二級結構間隔DNA以避免隨機序列造成DNA二級結構產生影響布朗運動值偵測上的誤差。SSB在單股DNA上的滑行遷移時有機會提供沒有SSB纏繞於單股DNA的區域，使RecA酵素能夠成核在上面，接著進行RecA的單體-單體的延長而形成核蛋白絲。我們證明EcRecA需要六個單位的RecA酵素在單股DNA上進行成核，而E38K突變體則僅需要少於六個單位的酵素以進行成核作用，後續的延長過程與核蛋白絲形成方式是相似的。

關鍵字

RecA ； RecA突變體 ； 單股DNA結合蛋白 ； 同源重組修復 ； 單分子拴球實驗

並列摘要

RecA proteins are essential for DNA homologous recombination and DNA repair in E. Coli and other eubacteria. In the typical DNA strand-exchange reaction, the RecA assembles on single-stranded DNA. For function in the DNA strand-exchange reaction, RecA have to first bind to single-stranded DNA to form nucleoprotein filaments. Single-stranded DNA generated in the cell during DNA metabolism is stablized and protected by binding of single-stranded DNA binding proteins. The formation of RecA filaments requires SSB protein to be displaced from the ssDNA. Project to monitor the SSB functioning as a barrier to RecA nucleation on ssDNA or RecA actively involves in the displacement of SSB from the ssDNA.
The mechanism of efficient SSB displacement by RecA was unclear given the tight binding of SSB to ssDNA. In order to comparing EcRecA assembly on ssDNA in the presence or absence of SSB have different function, we introduced a specific mutant RecA E38K encodes a form of RecA protein which is able to achieve the nucleation reaction without the help of RecA loading mediators RecFOR and exhibits constitutive SOS expression. We used single molecular tethered particle motion (TPM) techniques to directly observe the assembly dynamics of RecA proteins on individual DNA molecules by observing changes in Brownian motion (BM) produced by RecA binding. Without secondary structure gapped ssDNA substrates could exclude random sequence DNA forming secondary structure on BM detection inaccuracy. Diffusional migration of SSB on ssDNA provides the region for RecA nucleate on SSB-free ssDNA, then RecA proteins monomer-monomer extension on ssDNA to form RecA nucleoprotein filament. We demonstrated that EcRecA needs six subunits to nucleate on ssDNA, but E38K mutant only needs less than six subunits to nucleate. The extension function and nucleoprotein filament formation are similar both on EcRecA and E38K mutant.

並列關鍵字

RecA ； RecA mutant ； single-stranded DNA binding protein (SSB) ； homologous recombination ； single-molecule tethered particle motion

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