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 of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. 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-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.
Pingoud
V
V
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. vera.pingoud@chemie.bio.uni-giessen.de
Thole
H
H
Christ
F
F
Grindl
W
W
Wende
W
W
Pingoud
A
A
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010777:Photochemistry; D006180:Proton-Translocating ATPases; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae