Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure.
The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.
Lykke-Andersen
J
J
Institute of Molecular Biology, Copenhagen University, Denmark.
Garrett
R A
RA
Kjems
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics*; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; D010450:Endopeptidases