Investigating RecA filament formation of Escherichia coli and Deinococcus radiodurans along duplex DNA

憑藉著有效且精準的修復能力，抗輻射奇異球菌得以承受在強烈輻射照射或乾旱環境下所產生的數百個雙股DNA 斷裂，並完好存活。RecA 酵素在基因的重組與修復中，擁有非常重要且根本的地位。而抗輻射奇異球菌的RecA 酵素更是在目前所提出可能的基因修復機制中的各個步驟，都扮演關鍵的角色：一開始將損壞的DNA 延伸以及相互黏合的步驟，需要RecA 酵素的調節，接下來作用的其他酵素才能順利在雙股DNA 上進行反應。而眾多碎裂的片段在經過第一階段的修復後，形成較長的雙股DNA 片段，也需要透過RecA 酵素進行所謂的同源重組來完成修復。無論是哪個步驟，最重要的都是需要RecA 酵素結合雙股DNA，接續的反應才可能發生。因此在這個研究中，我們利用單分子栓球實驗，觀察每個雙股DNA 分子所接的球的布朗運動大小在加入RecA 酵素後隨著時間的變動情形，來研究RecA 酵素結合雙股DNA 的行為。我們發現，和一般最典型的細菌大腸桿菌比較起來，抗輻射奇異球菌可以非常快速的鬆開DNA 雙螺旋而結合雙股DNA，但在克服這個速率決定步驟之後結合上雙股DNA 的速度，卻比大腸桿菌慢。這種結合上雙股DNA 的方式，使抗輻射奇異球菌的RecA 酵素得以快速的結合在雙股DNA 的各個位置上，有效的進行全面的修復，反映了抗輻射奇異球菌對於面臨劇烈的基因損壞的修復模式。With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive the hundreds of double strand breaks inflicted by high doses of irradiation or desiccation. The RecA protein is essential for genome reconstitution. The RecA of Deinococcus radiodurans (DrRecA) plays role in both the early phase of repair categorized by an Extended synthesis-dependent strand annealing process (ESDSA) and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on dsDNA. We have developed single molecule tethered particle motion (TPM) protocols to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in Brownian motion produced by RecA binding. We demonstrate that DrRecA nucleation on dsDNA is much faster than observed with Escherichia coli RecA protein, but extension is slower. The DrRecA filament assembly mechanism reflects a cellular context in which large numbers of short RecA filaments are needed for global genomic repair