Bioprospecting reveals discrete structural modules governing strand-cleavage control and sequence preference in GIY-YIG nucleases
GIY-YIG nuclease domains (GIY-NDs) are widespread DNA endonucleases that generate single- ordouble-strand breaks, yet the structural basis by which they encode strand-cleavage control and sequence preference remains unclear. Here we combine phylogenetic bioprospecting, standardized fusion-based activity profiling, and structure-guided mutagenesis to define modular determinants of strand-cleavage behavior within the GIY-ND fold. Among 29 sequence-divergent GIY-NDs derived from homing endonucleases tested in a common heterologous architecture, nearly half retained catalytic activity despite truncation and recombination, underscoring the intrinsic modularity of the catalytic scaffold. Functional characterization of the intron-encoded homing endonuclease I-BamI revealed site specific bottom-strand nicking at a T-rich motif in its native genomic target, in contrast to the canonical CNNNG preference of I-TevI and the double-strand cleavage activity of all other characterized GIY-YIG homing endonucleases. Reciprocal sub-domain exchange between I-TevI and I-BamI localized control of second-strand cleavage to a-helix 2 (block 3), whereas a-helix 1 and its adjacent loop (block 2) governed sequence preference. Charge inversion at a single residue (R47) in block 3 suppressed second-strand cleavage about 4000-fold while preserving first-strand nicking, and combination with S48E produced complete conversion to a stable nickase state, establishing helix-proximal electrostatic polarity as a dominant regulator of strand-cleavage control. These findings demonstrate that strand selectivity and cleavage motif identity are mechanistically separable features encoded within a compact catalytic module, providing a framework for understanding GIY-YIG diversification and engineering of programmable nickase reagents.