The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage

Abstract

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138-Lys139 and Lys142-Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.

Figure 1.

Superposition of the bound and unbound DNA I-CreI structure. (a) Cα ribbon representation of I-CreI (pink) and the I-CreI-DNA (green) structures. DNA has been omitted for clarity. (b) Sequence alignment of the C-terminal region from members of the I-CreI family (42). The position of the mutated residues in the SKTRKT motif is indicated with a green triangle (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (c) Detailed view of S138, K139, K142 and T143 contacts with the DNA backbone. (d) Comparison of the positions of S138, K139, K142 and T143 between the bound (green) and unbound (pink) DNA structures.

Figure 1.

Superposition of the bound and unbound DNA I-CreI structure. (a) Cα ribbon representation of I-CreI (pink) and the I-CreI-DNA (green) structures. DNA has been omitted for clarity. (b) Sequence alignment of the C-terminal region from members of the I-CreI family (42). The position of the mutated residues in the SKTRKT motif is indicated with a green triangle (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (c) Detailed view of S138, K139, K142 and T143 contacts with the DNA backbone. (d) Comparison of the positions of S138, K139, K142 and T143 between the bound (green) and unbound (pink) DNA structures.

Figure 2.

Electrophoretic mobility shift assays of the C-terminal truncated, double and single mutants in the presence of Mg²⁺ and Ca²⁺ (Gels with the experimental data for the K_D measurements are available as Supplementary Data).

Figure 3.

Summary of the gel in vitro cleavage assay of the C-terminal truncated, double and single mutants. The gels with the experimental data for all the cleavage experiments are available as Supplementary Data.

Figure 4.

Profiling of single mutants. (a) Yeast screening assay principle. A strain harboring the expression vector encoding a single mutant is mated with a strain harboring a reporter plasmid. In the reporter plasmid, a LacZ reporter gene is interrupted with an insert containing one of the target sites of interest, flanked by two direct repeats. Upon mating, the meganuclease (gray oval) generates a double-strand break at the site of interest, allowing restoration of a functional LacZ gene by single-strand annealing (SSA) between the two flanking direct repeats. (b) DNA targets. The 10AAA_5GTC_P target (top) is a palindrome cleaved by I-CreI. The different name (The 10AAA_P or 5GTC_P) indicates whether it was used in the 5NNN_P or 10NNN_P experiment. All targets used in this study are palindromes derived from 10AAA_5GTC_P by substitution of six nucleotides in ±8, ±9 and ±10 or in ±3, ±4 and ±5. A few examples are shown (bottom). The 10GGG_P target differs from the 10AAA_P target by the GGG triplet in –10, –9, –8 and CCC in 8, 9, 10, etc. The 5GGG_P target differs from 10AAA_5GTC_P by the GGG triplet in −5, −4, −3 and CCC in 3, 4, 5, etc. (c) Mutant target profiles. Each mutant was profiled in yeast on a series of 128 palindromic targets (5NNN_P or 10NNN_P). An example of cleavage activity of 15 targets in yeast is presented for a single mutant (K139M) and I-CreI D75N. As described previously (11), blue staining indicates cleavage. Additionally a representation of the cleavage profile of all single mutants compared to I-CreI D75N and I-CreI. Gray levels reflect the intensity of the signal. I-CreI is toxic in yeast (11) and profiles have been established at 30°C instead of 37°C. All other mutants were studied at 37°C.