Modular determinants of cleavage preference in GIY-YIG nucleases revealed by block-based DNA shuffling and directed evolution.

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 cle

SUBMITTER: Loedige KW 

PROVIDER: S-EPMC12774661 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature