ArticleNucleic acids research2026
Modular determinants of cleavage preference in GIY-YIG nucleases revealed by block-based DNA shuffling and directed evolution.
Article in Nucleic acids research, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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1 citing paper in PubMed.
- A Modified Methyl Transferase Cofactor to Selectively Silence Gene Expression in Escherichia coli.Chembiochem : a European journal of chemical biology · 2026Article
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5 authors.
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Abstract
GIY-YIG homing endonucleases are mobile genetic elements found in phage, bacterial, and organellar genomes. Their modular architecture and sequence-tolerant DNA cleavage properties likely represent evolutionary adaptations to tolerate genetic drift at target sites and enable target-site switching. To investigate modular determinants of GIY-YIG nuclease cleavage preference, we constructed 128 chimeric nucleases by shuffling structural blocks between the prototypical GIY-YIG homing endonuclease I-TevI (CNNNG motif preference) and its isoschizomer I-BmoI (NNNNG preference). Chimeras containing a swapped $\alpha$-helix1 and adjacent loop exhibited altered motif preferences, highlighting this region as a modular determinant, whereas swaps in other regions disrupted activity without altering cleavage preference. Directed evolution of nonconserved residues within this region identified a cluster (R30, K33, E36, C39) where substitutions enabled cleavage of targets poorly recognized by wild-type I-TevI, including variants with reprogrammed preference toward TNNNG and GNNNG motifs. Our findings define a modular and structural basis for DNA cleavage preference in the GIY-YIG nuclease domain and suggest that recombination of structural subunits could accelerate adaptation to new target sites over evolutionary time scales. These findings further support a strategy for engineering GIY-YIG nuclease domains with expanded cleavage motif selectivity.
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