Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.
Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved 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 unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential 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 endonucleases 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 motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.
Lykke-Andersen
J
J
RNA Regulation Centre and Institute of Molecular Biology, Copenhagen University, Denmark.
Garrett
R A
RA
Kjems
J
J
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism; D017665:Hydroxyl Radical; D007438:Introns / Q000235:genetics*; D007501:Iron / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D005296:Ferrous Compounds; D011993:Recombinant Fusion Proteins; D017665:Hydroxyl Radical; D004247:DNA; D007501:Iron; D004706:Endodeoxyribonucleases; C106900:endodeoxyribonuclease I-PorI; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific; D008274:Magnesium