IDENTIFICATION OF LYS-403 IN THE PI-SCEI HOMING ENDONUCLEASE AS PART OF A SYMMETRICAL CATALYTIC CENTER

Superposition of the PI-SceI and I-CreI homing endonuclease three-dime nsional x-ray structures indicates general similarity between the I-Cr eI homodimer and the PI-SceI endonuclease domain. Saddle-shaped struct ures 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 reactio n pathway was reported previously. Here, we demonstrate the significan ce of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has lit tle effect on the DNA binding activity of the mutant enzyme. Substitut ion 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 protei ns with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homin g endonucleases suggests that these enzymes use a common reaction mech anism to cleave double-stranded DNA.