Ito Kentaro, Argunhan Bilge, Tsubouchi Hideo, Iwasaki Hiroshi
Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology.
Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology;
J Vis Exp. 2019 Feb 13(144). doi: 10.3791/59073.
The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.
由Rad51介导的DNA链交换反应是同源重组的关键步骤。在该反应中，Rad51在单链DNA（ssDNA）上形成核蛋白丝，并非特异性地捕获双链DNA（dsDNA）以寻找同源序列。遇到同源序列后，Rad51催化DNA链交换，介导ssDNA与dsDNA的互补链配对。该反应在体内受到多种辅助蛋白的高度调控。尽管传统的生化分析已成功用于体外研究此类辅助蛋白的作用，但由于反应中间体的不稳定和短暂性，对中间体形成及其向最终产物进展的动力学分析已证明具有挑战性。为了在溶液中直接观察这些反应步骤，建立了基于荧光共振能量转移（FRET）的该反应实时观察系统。实时观察的动力学分析表明，由Rad51介导的DNA链交换反应遵循一个三步反应模型，包括形成三链DNA中间体、该中间体的成熟以及单链DNA从成熟中间体的释放。Swi5-Sfr1复合物是一种在真核生物中保守的辅助蛋白，强烈增强该反应的第二步和第三步。本文介绍的基于FRET的分析使我们能够揭示重组辅助蛋白刺激Rad51的DNA链交换活性的分子机制。本方案的主要目标是增加同源重组领域研究人员可用的技术种类，特别是那些研究粟酒裂殖酵母以外物种蛋白质的人员，以便确定本文所述发现的进化保守性。
