Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site.
Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.
Ellison
E L
EL
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Vogt
V M
VM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D016172:DNA Fingerprinting; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D010804:Physarum / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation
D004271:DNA, Fungal; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease