MAPPING METAL-IONS AT THE CATALYTIC CENTERS OF 2 INTRON-ENCODED ENDONUCLEASES

Divalent metal ions play a crucial role in forming the catalytic centr es of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yiel ded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites, Specific hydroxyl radica l-induced cleavage mas observed within, and immediately after two cons erved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unabl e to support endonucleolytic activity, was performed to distinguish be tween the individual metal ions in the complex. Mutations of single am ino acids in this region impaired catalytic activity and caused the pr eferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonuc leases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG moths, respectively. The dual pr otein/nucleic acid footprinting approach described here is generally a pplicable to other protein-nucleic acid complexes when the natural met al ion can be replaced by Fe2+.