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

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

Author information

1
Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
2
Yale University, New Haven, CT, United States.
3
Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. Electronic address: hongweiwang@tsinghua.edu.cn.

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

PMID:
29458758
DOI:
10.1016/bs.mie.2017.12.002
[Indexed for MEDLINE]

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