Subramanyam Shyamal, Kinz-Thompson Colin D, Gonzalez Ruben L, Spies Maria
University of Iowa Carver College of Medicine, Iowa City, IA, United States.
Columbia University, New York, NY, United States.
Methods Enzymol. 2018;600:201-232. doi: 10.1016/bs.mie.2017.12.008. Epub 2018 Feb 1.
Human RAD51 promotes accurate DNA repair by homologous recombination and is involved in protection and repair of damaged DNA replication forks. The active species of RAD51 and related recombinases in all organisms is a nucleoprotein filament assembled on single-stranded DNA (ssDNA). The formation of a nucleoprotein filament competent for the recombination reaction, or for DNA replication support, is a delicate and strictly regulated process, which occurs through filament nucleation followed by filament extension. The rates of these two phases of filament formation define the capacity of RAD51 to compete with the ssDNA-binding protein RPA, as well as the lengths of the resulting filament segments. Single-molecule approaches can provide a wealth of quantitative information on the kinetics of RAD51 nucleoprotein filament assembly, internal dynamics, and disassembly. In this chapter, we describe how to set up a single-molecule total internal reflection fluorescence microscopy experiment to monitor the initial steps of RAD51 nucleoprotein filament formation in real-time and at single-monomer resolution. This approach is based on the unique, stretched-ssDNA conformation within the recombinase nucleoprotein filament and follows the efficiency of Förster resonance energy transfer (E) between two DNA-conjugated fluorophores. We will discuss the practical aspects of the experimental setup, extraction of the FRET trajectories, and how to analyze and interpret the data to obtain information on RAD51 nucleation kinetics, the mechanism of nucleation, and the oligomeric species involved in filament formation.
人类RAD51通过同源重组促进准确的DNA修复，并参与受损DNA复制叉的保护和修复。所有生物体中RAD51及相关重组酶的活性形式是组装在单链DNA（ssDNA）上的核蛋白丝。形成能够进行重组反应或支持DNA复制的核蛋白丝是一个微妙且受到严格调控的过程，它通过丝状物成核，随后是丝状物延伸来发生。丝状物形成的这两个阶段的速率决定了RAD51与单链DNA结合蛋白RPA竞争的能力，以及所形成的丝状物片段的长度。单分子方法可以提供关于RAD51核蛋白丝组装动力学、内部动力学和解聚的大量定量信息。在本章中，我们描述了如何建立一个单分子全内反射荧光显微镜实验，以实时且以单单体分辨率监测RAD51核蛋白丝形成的初始步骤。这种方法基于重组酶核蛋白丝内独特的拉伸单链DNA构象，并跟踪两个与DNA结合的荧光团之间的Förster共振能量转移（E）效率。我们将讨论实验设置的实际方面、FRET轨迹的提取，以及如何分析和解释数据以获得关于RAD51成核动力学、成核机制以及参与丝状物形成的寡聚体种类的信息。
