A model for the PI-SceIxDNA complex based on multiple base and phosphate backbone-specific photocross-links.
We have synthesized different oligodeoxynucleotides carrying, in single positions of the >36 bp recognition site of PI-SceI, photoreactive base analogues (5-iododeoxypyrimidines) or phosphate modifications (p-azidophenacylphosphorothioates) and used them in photocross-linking experiments with PI-SceI to probe the protein-DNA interface of the specific complex between the homing endonuclease PI-SceI and its DNA substrate. One base-specific and several backbone-specific cross-links were analyzed in detail: the cross-linking positions were identified by Edman degradation of isolated cross-linked peptidexoligodeoxynucleotide adducts and confirmed by site-directed mutagenesis. Based on these results and the crystal structure of PI-SceI, a model for the structure of the PI-SceIxDNA complex is proposed.
Christ
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F
Institut für Biochemie Fachbereich 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
Steuer
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Thole
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H
Wende
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W
Pingoud
A
A
Pingoud
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D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae