Mapping of a DNA binding region of the PI-SceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucle ases that is generated by a protein splicing reaction. PI-SceI has a bipart ite domain structure, and the protein splicing and endonucleolytic reaction s are catalyzed by residues in domains I and II, respectively. Structural a nd mutational evidence indicates that both domains mediate DNA binding. Tre atment of the protein with trypsin breaks a peptide bond within a disordere d region of the endonuclease domain situated between residues Val-270 and L eu-280 and interferes with the ability of this domain to bind DNA. To ident ify specific residues in this region that are involved in DNA binding and/o r catalysis, alanine-scanning mutagenesis was used to create a set of PI-Sc eI mutant proteins that were assayed for activity. One of these mutants, N2 81A, was >300-fold less active than wild-type PI-SceI, and two other protei ns, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonucleas e domains of these mutant proteins. We mapped the approximate position of t he disordered region to one of the ends of the 31 base pair PI-SceI recogni tion sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI- SceI docked to its DNA substrate that suggests that one or more residues id entified here are responsible for contacting base pair A/T-9, which is esse ntial for substrate binding.