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Updated: Jun 9, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Published on: February 7, 2021
Folding, DNA recognition, and function of GIY-YIG endonucleases: crystal structures of R.Eco29kI
Amanda Nga-Sze Mak1, Abigail R Lambert, Barry L Stoddard
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Abstract:
The GIY-YIG endonuclease family comprises hundreds of diverse proteins and a multitude of functions; none have been visualized bound to DNA. The structure of the GIY-YIG restriction endonuclease R.Eco29kI has been solved both alone and bound to its target site. The protein displays a domain-swapped homodimeric structure with several extended surface loops encircling the DNA. Only three side chains from each protein subunit contact DNA bases, two directly and one via a bridging solvent molecule. Both tyrosine residues within the GIY-YIG motif are positioned in the catalytic center near a putative nucleophilic water; the remainder of the active site resembles the HNH endonuclease family. The structure illustrates how the GIY-YIG scaffold has been adapted for the highly specific recognition of a DNA restriction site, in contrast to nonspecific DNA cleavage by GIY-YIG domains in homing endonucleases or structure-specific cleavage by DNA repair enzymes such as UvrC.
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