Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

Abstract

The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional 'artificial' mutations, to optimize its in vivo activity.

Figure 1.

Isolation of optimized I-AniI variants by a bacteria based cleavage assay. (A) The expression of the toxic gene is restricted on the selective plates. WT I-SceI significantly cleaved its wild type target site in bacteria, but WT I-AniI inefficiently did against its physiological (WT I-AniI) and optimal (LIB4) sequences. The Y2 and M5 variants improved the cleavage activity. (B) Ribbon model of WT I-AniI structure. Gray sticks indicates the physiological target DNA. Mutated residues in Y2 construct are labeled in red; remaining mutated residues in M5 construct are labeled in blue.

Figure 2.

Sequence alignment of I-AniI homologs. The alignment was performed using CLUSTALW program. Open triangles indicate positions of F13Y and S111Y in the Y2 I-AniI construct. AAX39426, ABC86634, ABU49435, AAX34413, AAB95258 and bI3 maturase are LAGLIDADG proteins from Neosartorya fischeri, Gibberella zeae, Phaeosphaeria nodorum SN15, Agrocybe chaxingu, Venturia inaequalis and Saccharomyces cerevisiae, respectively. Closed triangles represent sites of additional mutations I55V, F91I and S92T in the M5 I-AniI construct. Asterisks indicate remaining positions of point mutations isolated in the cleavage activity screen as listed in Table 1.

Figure 3.

Secondary structure, thermal stability and thermal activity profile analyses of WT and mutant I-AniI variants. (A) Circular dichroism analysis. Far-UV CD spectra were measured between 190 and 260 nm at 25°C. (B) Temperature-dependent denaturation on the basis of changes in circular dichroism ellipticity at 222 nm. Temperature of 50% transition was 53.9, 52.6 and 43.5°C for WT, Y2 variant and M5 variant, respectively. (C) Thermal profiles of the cleavage activity. The reactions ran at 22, 30, 37, 42, 47, 55 and 65°C. Relative activity was shown by taking cleavage activity at 47°C as 100%. Errors mean ± S.D.

Figure 4.

DNA binding affinity, DNA cleavage activity and intron splicing activity of WT and ‘Y2’ optimized I-AniI variant. (A) Binding assay with WT I-AniI (left) and Y2 variant (right) against WT I-AniI site by isothermal titration calorimetry. The left graph is taken from our previous report (9). Dissociation constants and thermodynamic values are summarized in Supplementary Table S1. (B) In vitro cleavage assay against the WT I-AniI target site. A serial dilution of each I-AniI variant was incubated with PCR fragment containing a single copy of the target site at 37°C. The reaction products were quantified using the NIH ImageJ program. Errors mean ± S.D. Sub: linearized plasmid substrate; prod: cleaved products. (C) Relative cleavage of top and bottom DNA strands by WT and Y2 I-AniI. The top strand includes the coding sequence of the mitochondrial apocytochrome B gene from Aspergillus nidulans (5′-TGAGGAGGATTTCTCTGAAA-3′). Sub = substrate; Prod = product. Open ovals = WT, filled ovals = Y2. The data are fit to an equation that describes a first order reaction (see Materials and Methods section). Note that the Y2 variant cleaves the bottom strand more rapidly that the WT protein. For values, see Supplementary Table S2. The designation of ‘top’ and ‘bottom’ strand in these methods is opposite that previously described in (22) where the labelling of strands and cleavage products were inadvertantly reversed. (D) Relative intron splicing activity of WT and Y2 I-AniI. Pre = precursor RNA (substrate); I3E = splicing intermediate after 5′-splice site cleavage with the 3′-exon still linked to the intron; I = free intron (product). The ligated exon product is not shown. The asterisk demarcates an RNA degradation product. Open ovals = WT; filled ovals = Y2. The data are fit to an equation that describes a first order reaction (see Materials and Methods section). For values, see Supplementary Table S2.

Figure 5.

Comparative in vivo gene conversion activity of WT I-AniI and the Y2 variant. (A) Schematic representation of the DR-GFP reporter plasmid. The reporter plasmid harbors two non-functional copies of the GFP gene, one interrupted by two stop codons followed by a target site for either I-AniI or I-SceI, the other encoding the 5′- and 3′-truncated gene. (B) Representative FACS plot of gene conversion on the DR-GFP reporter with WT AniI site (I-AniI DR-GFP). 293T cells were transiently co-transfected with the reporter and an expression plasmid for either WT I-AniI or Y2 variant, and analyzed by flow cytometry at 48 hours post transfection. In each plot, the fluorescent side scatter ‘SSC’ (as a standard of cell viability) is plotted on the Y axis and the GFP-associated fluorescent signal ‘GFP’ (a measurement of recombiantion of the reporter plasmid in the transfected cells) is plotted on the X-axis. (C,D) graph depicting percent gene conversion of I-AniI DR-GFP (C) and I-SceI DR-GFP (D) induced by either WT I-AniI, Y2 variant or I-SceI. Data represents three independent experiments performed in duplicate normalized to observed background by transfection of the DR-GFP reporter alone, Errors mean + SE. (E) Expression of HEs in the transfected cells. The expression level was analyzed by Western blotting using an antibody against hemagglutinin (HA)-epitope (upper) and β-actin as a control (lower).

Figure 6.

Structural analysis of the Y2 I-AniI construct. Electron density maps for several of the structural features shown here are provided in Supplementary Figure S3. For clarity of viewing, the orientation of the structure shown in panels B–G are rotated 180°C relative to the canonical orientation of the DNA substrate sequence shown in panel A. (A) Sequence of the DNA duplex used for the crystallization. The target site (20 basepairs) is numbered from positions −10 to +10, relative to the center of the four-base overhangs generated by DNA cleavage. The strand breaks are introduced between positions +2 and +3 in the top strand, and between −2 and −3 in the bottom strand. (B) Y2 I-AniI (cyan) in complex with the DNA duplex (grey) superimposed against WT I-AniI (light yellow) bound to the same DNA target sequence (white) (PDB: 2QOJ). The extended loop of Y2 I-AniI linking the N-terminal and the C-terminal domains is shown in dark blue. The arrow in panel B and C points towards S111Y. (C) Magnification of region surrounding residue 111 (S111Y mutation in the Y2 I-AniI construct). (D) Magnification of region surrounding residue 120. (E) Magnification of region surrounding residue 94 and its surrounding active site residues and DNA bases. (F) Magnification of region surrounding basepairs −3 and −4. (G) Magnification of region surrounding residue 13 (F13Y in the Y2 I-AniI construct).