Single-Molecule Analysis of Replication Protein A–DNA Interactions

Abstract 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.