Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed to bind DNA. At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously. Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme. Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity. Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|