Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51
Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51
复制标题
DOI：
10.3791/59073
复制
发表时间：
2019-02-01
影响因子：
1.2
通讯作者：
Iwasaki, Hiroshi
中科院分区：
文献类型：
--
作者：
Ito, Kentaro;Argunhan, Bilge;Iwasaki, Hiroshi
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，并非特异性捕获双链DNA （dsDNA）以询问其同源序列。在遇到同源性后，Rad51催化DNA链交换，介导ssDNA与dsDNA互补链的配对。该反应在体内受到许多辅助蛋白的高度调控。尽管传统的生化分析已经成功地用于检测这种辅助蛋白在体外的作用，但由于反应中间体的不稳定性和短暂性，中间体形成及其向最终产物发展的动力学分析已被证明具有挑战性。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，Rad51介导的DNA链交换反应遵循三步反应模型，包括三链DNA中间体的形成、该中间体的成熟以及成熟中间体释放ssDNA。Swi5-Sfr1复合体是一种在真核生物中保守的辅助蛋白，它强烈地促进了该反应的第二步和第三步。本文提出的基于fret的分析使我们能够揭示重组辅助蛋白刺激Rad51 DNA链交换活性的分子机制。本协议的主要目标是增强同源重组领域的研究人员可用的技术储备，特别是那些与除裂糖菌以外的其他物种的蛋白质一起工作的技术，以便确定本文所提出的发现的进化守恒性。
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.