Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease.
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.
McConnell Smith
Audrey
A
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, North Seattle, WA 98109, USA.
Takeuchi
Ryo
R
Pellenz
Stefan
S
Davis
Luther
L
Maizels
Nancy
N
Monnat
Raymond J
RJ
Stoddard
Barry L
BL
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
D012194:RNA-Directed DNA Polymerase; C454879:maturase I-AniI; D004720:Endonucleases; D003850:Deoxyribonuclease I