A model for the PI-Scel x DNA complex based on mutiple base and phosphate backbone-specific photocross-links

We have synthesized different oligodeoxynucleotides carrying, in single pos itions of the >36 bp recognition site of PI-SceI, photoreactive base analog ues (5-iododeoxypyrimidines) or phosphate modifications (p-azidophenacylpho sphorothioates) and used them in photocross-linking experiments with PI-Sce I to probe the protein-DNA interface of the specific complex between the ho ming endonuclease PI-SceI and its DNA substrate. One base-specific and seve ral backbone-specific cross-links were analyzed in detail: the cross-linkin g positions were identified by Edman degradation of isolated cross-linked p eptide x oligodeoxynucleotide adducts and confirmed by site-directed mutage nesis. Based on these results and the crystal structure of PI-SceI, a model for the structure of the PI-SceI x DNA complex is proposed. (C) 2000 Acade mic Press.