Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference

• Published in: Nucleic acids research Vol. 44; no. 15; pp. 7350 - 7359
• Main Authors: Roy, Alexander C, Wilson, Geoffrey G, Edgell, David R
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 06.09.2016
• Subjects: Amino Acid Substitution - genetics; Directed Molecular Evolution; DNA Cleavage; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Introns - genetics; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Nucleic Acid Enzymes; Protein Domains - genetics; Structure-Activity Relationship; Substrate Specificity
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkw614

Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.