Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease - PubMed (original) (raw)

Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease

Audrey McConnell Smith et al. Proc Natl Acad Sci U S A. 2009.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Point mutations generate an I-AniI nickase. (A) (Upper) Ribbon diagram of wild-type I-AniI (α-helices, green; β-sheets, blue) bound to its DNA target site (gray). (Lower) Attack on the DNA backbone mediated by residues Q171 and K227, in the periphery of the right active site. (B) Cleavage of a plasmid substrate containing the Lib4 I-AniI target sequence by wild-type, Q171K, K227M, and Q171K/K227M I-AniI variants at 10, 100, and1000 nM enzyme. Digestion with I-HmuI provides a nicked substrate control; uncut plasmid and EcoRI-linearized plasmid are at the right. The box indicates the strongest nickase, K227M I-AniI. (C) Gel electrophoresis of products of cleavage of a 5′ end-labeled 313-bp duplex substrate (Left, asterisk, 32P-label) by wild-type I-AniI, I-AniI K227M, and Y2 I-AniI K227M. Predicted radiolabeled fragments of 254 and 63 nucleotides are indicated.

Fig. 2.

DNA target site cleavage and nicking by I-AniI and I-AniI nickase. (A) Time course of DNA digestion by I-AniI, assayed at 10-min intervals over 2 h. (B) Time course of DNA digestion by I-AniI nickase, assayed at 0, 0.5, 1, 2, 3, 4, 7.5, 10, 15, 20, 25, and 30 min, and then at 10-min intervals up to 90 min. (C) Plot of reaction time versus percentage cleavage or nicking. Curves fit with program Prism5: nicking _t_1/2 = 7.6 min, cleavage _t_1/2 = 59.6 min.

Fig. 3.

Specificity profile of I-AniI nickase. Fraction of nicked plasmid substrate recovered is shown for each of 60 targets assayed; indicated colors denote sequence at each position. The bold horizonal line illustrates the relative cleavage of the wild-type target site, measured as a control experiment during each set of digests. Examples of digests are shown in

Fig. S4

Fig. 4.

In vivo recombination stimulated by I-AniI cleavase or nickase. (A) Reporter assay for recombination in cis. Recombination of an inactive 3′-GFP gene (GFPi) with a cleaved 5′-GFPi allele generates a functional GFP+ gene. Gray arrows, GFP alleles; filled box with white X, I-AniI cleavage site and stop codon; arrow, promoter; PuroR, selectable puromycin resistance marker. (B) Reporter assay for recombination in trans. Recombination of a chromosomally integrated target with a transfected repair template (GFPt) generates a functional GFP+ gene. GFP+ alleles and cells in both assays are detected and quantified by flow cytometry. Other notations are as in A. (C) Fold increase in the frequency of GFP+ cells in the cis assay shown in A above, relative to reporter-only background, after expression of Y2 I-AniI cleavase (c), Y2 I-AniI nickase (n), or catalytically inactive Y2 I-AniI nickase (d). Additional primary data are presented in

Table S1

. (D) Fold increase in the frequency of GFP+ cells in the trans assay shown in B above, relative to reporter-only background, after expression of Y2 I-AniI cleavase (c), Y2 I-AniI nickase (n), or a catalytically inactive form of Y2 I-AniI nickase (d). Additional primary data are presented in

Table S1

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Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease - PubMed (original) (raw)