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

Lingyun Zhao, Jingfei Xu, Weixing Zhao, Patrick Sung, Hong Wei Wang

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages179-199
Number of pages21
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume600
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Cryo-EM
  • Helical reconstruction
  • High resolution
  • Homologous recombination
  • Human RAD51

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Zhao, L., Xu, J., Zhao, W., Sung, P., & Wang, H. W. (2018). Determining the RAD51-DNA Nucleoprotein Filament Structure and Function by Cryo-Electron Microscopy. In Methods in Enzymology (pp. 179-199). (Methods in Enzymology; Vol. 600). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2017.12.002