title: Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference.
abstract: 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.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Roy
authors.affiliation: New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Wilson
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada dedgell@uwo.ca.
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D053837:DNA Cleavage; D019020:Directed Molecular Evolution; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007438:Introns / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D000072417:Protein Domains / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease; C065074:I-TEVI endonuclease