Single-Molecule Analysis of Replication Protein A-DNA Interactions

Methods Enzymol. 2018:600:439-461. doi: 10.1016/bs.mie.2017.11.016.

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.

Keywords: Biotin labeling; Fluorescent protein labeling; RPA; Replication protein A; Single-stranded DNA binding; TIRFM.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Fluorescent Dyes / chemistry
  • Image Processing, Computer-Assisted / methods
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinational DNA Repair
  • Replication Protein A / chemistry
  • Replication Protein A / isolation & purification
  • Replication Protein A / metabolism*
  • Single Molecule Imaging / instrumentation
  • Single Molecule Imaging / methods*
  • Staining and Labeling / methods
  • Video Recording / instrumentation
  • Video Recording / methods

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Fluorescent Dyes
  • RPA1 protein, human
  • RPA3 protein, human
  • Recombinant Proteins
  • Replication Protein A
  • RPA2 protein, human