Chemical probing shows that the intron-encoded endonuclease I-SceI distorts DNA through binding in monomeric form to its homing site.
Despite its small size (27.6 kDa), the group I intron-encoded I-SceI endonuclease initiates intron homing by recognizing and specifically cleaving a large intronless DNA sequence. Here, we used gel shift assays and footprinting experiments to analyze the interaction between I-SceI and its target. I-SceI was found to bind to its substrate in monomeric form. Footprinting using DNase I, hydroxyl radical, phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and base-modifying reagents revealed the asymmetric nature of the interaction and provided a first glimpse into the architecture of the complex. The protein interacts in the minor and major grooves and distorts DNA at three distinct sites: one at the intron insertion site and the other two, respectively, downstream (-8, -9) and upstream (+9, +10) from this site. The protein appears to stabilize the DNA curved around it by bridging the minor groove on one face of the helix. The scissile phosphates would lie on the outside of the bend, facing in the same direction relative to the DNA helical axis, as expected for an endonuclease that generates 3' overhangs. An internally consistent model is proposed in which the protein would take advantage of the concerted flexibility of the DNA sequence to induce a synergistic binding/kinking process, resulting in the correct positioning of the enzyme active site.
Laboratoire de Physique et Chimie Biomoléculaires, Institut Curie, Rue des Saint-Pères, Paris, France.
B
B
Beylot
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A
Spassky
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D017665:Hydroxyl Radical; D007438:Introns; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D017665:Hydroxyl Radical; D004247:DNA; C037725:SCEI protein, S cerevisiae; D003850:Deoxyribonuclease I; D015252:Deoxyribonucleases, Type II Site-Specific