Determining the RAD51-DNA Nucleoprotein Filament Structure and Function by Cryo-Electron Microscopy

Methods Enzymol. 2018:600:179-199. doi: 10.1016/bs.mie.2017.12.002. Epub 2018 Feb 1.

Abstract

Homologous recombination is a universal tool for DNA double-strand break and replication fork repair, and it is catalyzed by a highly conserved family of recombinases. In eukaryotes, Rad51 is the recombinase that catalyzes the pairing of homologous DNA molecules and the exchange of strands between the paired molecules. Rad51 assembles on single-stranded DNA (ssDNA) stemming from lesion processing to form a right-handed helical polymer that engages then samples double-stranded DNA (dsDNA) for homology. Upon matching with a homologous sequence, the Rad51-bound ssDNA invades the dsDNA, leading to the formation of a DNA joint with concomitant displacement of the strand of like polarity. The Rad51-DNA filaments are amenable to structural studies using cryo-electron microscopy (cryo-EM). In particular, recent technical breakthroughs in cryo-EM have made it possible to define the structure and function of human RAD51 at near-atomic resolution. In this chapter, we describe our cryo-EM approach to capture the human RAD51 filament structures in various stages of catalysis. The approach may also be useful for related recombinases and other helical assemblies.

Keywords: Cryo-EM; Helical reconstruction; High resolution; Homologous recombination; Human RAD51.

Publication types

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

MeSH terms

  • Cryoelectron Microscopy / instrumentation
  • Cryoelectron Microscopy / methods*
  • DNA Breaks, Double-Stranded
  • DNA, Single-Stranded / isolation & purification
  • DNA, Single-Stranded / metabolism
  • DNA, Single-Stranded / ultrastructure*
  • Humans
  • Molecular Docking Simulation
  • Nucleoproteins / isolation & purification
  • Nucleoproteins / metabolism
  • Nucleoproteins / ultrastructure*
  • Rad51 Recombinase / isolation & purification
  • Rad51 Recombinase / metabolism
  • Rad51 Recombinase / ultrastructure*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Recombinational DNA Repair*

Substances

  • DNA, Single-Stranded
  • Nucleoproteins
  • Recombinant Proteins
  • RAD51 protein, human
  • Rad51 Recombinase