Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family o f homing endonucleases, According to the crystal structure and mutational s tudies, this endonuclease consists of two domains, one responsible for prot ein splicing, the other for DNA cleavage, and both presumably for DNA bindi ng, To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA, Sixty-three double-str anded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions o f the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm), The best cross-linking yie ld (approximately 30%) was obtained with an oligodeoxynucleotide with a B-i ododeoxyuridine at position +9 in the bottom strand, The subsequent analysi s showed that cross-linking had occurred with amino acid His-333, 6 amino a cids after the second LAGLIDADG motif, With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linkin g was observed, indicating the specificity of the crosslinking reaction. Ch emical modification of His residues in PI-SceI by diethylpyrocarbonate lead s to a substantial reduction in the binding and cleavage activity of PI-Sce I, This inactivation can be suppressed by substrate binding. This result fu rther supports the finding that at least one His residue is in close contac t to the DNA, Based on these and published results, conclusions are drawn r egarding the DNA binding site of PI-SceI.