Bain Fletcher E, Fischer Laura A, Chen Ran, Wold Marc S
University of Iowa Carver College of Medicine, Iowa City, IA, United States.
Washington University, St. Louis, MO, United States.
Methods Enzymol. 2018;600:439-461. doi: 10.1016/bs.mie.2017.11.016.
Replication protein A (RPA) is a highly conserved, eukaryotic ssDNA-binding protein essential for genome stability. RPA interacts with ssDNA and with protein partners to coordinate DNA replication, repair, and recombination. Single-molecule analysis of RPA-DNA interactions is leading to a better understanding of the molecular interactions and dynamics responsible for RPA function in cells. Here, we first describe how to express, purify, and label RPA. We then describe how to prepare materials and carry out single-molecule experiments examining RPA-DNA interactions using total internal reflection fluorescence microscopy (TIRFM). Finally, the last section describes how to analyze TIRFM data. This chapter will focus on human RPA. However, these methods can be applied to RPA homologs from other species.
复制蛋白A（RPA）是一种高度保守的真核单链DNA结合蛋白，对基因组稳定性至关重要。RPA与单链DNA以及蛋白质伙伴相互作用，以协调DNA复制、修复和重组。对RPA-DNA相互作用的单分子分析有助于更好地理解细胞中负责RPA功能的分子相互作用和动力学。在这里，我们首先描述如何表达、纯化和标记RPA。然后我们描述如何制备材料并使用全内反射荧光显微镜（TIRFM）进行检测RPA-DNA相互作用的单分子实验。最后，最后一部分描述如何分析TIRFM数据。本章将重点介绍人类RPA。然而，这些方法也可应用于其他物种的RPA同源物。
