title: Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease.
abstract: Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at approximately 1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, North Seattle, WA 98109, USA.
authors.forename: Audrey
authors.initials: A
authors.lastname: McConnell Smith
authors.forename: Ryo
authors.initials: R
authors.lastname: Takeuchi
authors.forename: Stefan
authors.initials: S
authors.lastname: Pellenz
authors.forename: Luther
authors.initials: L
authors.lastname: Davis
authors.forename: Nancy
authors.initials: N
authors.lastname: Maizels
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Monnat
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain; D053837:DNA Cleavage; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D012194:RNA-Directed DNA Polymerase; C454879:maturase I-AniI; D004720:Endonucleases; D003850:Deoxyribonuclease I