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A novel endonuclease mechanism directly visualized for I-PpoI
E A Galburt1, B Chevalier, W Tang
1Fred Hutchinson Cancer Research Center and the Graduate Programs in Molecular and Cell Biology and Biomolecular Structure and Design, University of Washington, 1100 Fairview Ave. N. A3-023, Seattle, Washington 98109, USA.
Abstract:
A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.