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A novel endonuclease mechanism directly visualized for I-PpoI

Nature Structural Biology volume 6, pages 1096–1099 (1999)Cite this article

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.

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Figure 1: Electron density difference maps for three catalytic complexes formed by I-PpoI.
Figure 2: Anomalous difference Fourier map of I-PpoI and DNA with bound manganese ion, shown in the same orientation as Fig.1.
Figure 3: Interactions of DNA, solvent and bound cation in the region of the scissile phosphate.
Figure 4: Proposed catalytic mechanism for the I-PpoI/Serratia endonucleases.

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Authors and Affiliations

  1. Fred 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, 98109, Washington, USA

    Eric A. Galburt, Brett Chevalier, Melissa S. Jurica, Karen E. Flick & Barry L. Stoddard

  2. Department of Pathology, University of Washington, Box 357705, Seattle, 98195, Washington, USA

    Weiliang Tang & Raymond J. Monnat Jr.

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  1. Eric A. Galburt
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  2. Brett Chevalier
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  3. Weiliang Tang
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Correspondence to Barry L. Stoddard.

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Galburt, E., Chevalier, B., Tang, W. et al. A novel endonuclease mechanism directly visualized for I-PpoI. Nat Struct Mol Biol 6, 1096–1099 (1999). https://doi.org/10.1038/70027

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