title: Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.
abstract: LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.
authors.affiliation: Program in Molecular and Cellular Biology, University of Washington, Box 357275, Seattle, WA 98195, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Jacoby
authors.forename: Michael
authors.initials: M
authors.lastname: Metzger
authors.forename: Betty W
authors.initials: BW
authors.lastname: Shen
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
mesh_terms: D000595:Amino Acid Sequence; D003461:Crystallography; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004706:Endodeoxyribonucleases