Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

Jonathan M. Hadden, Anne-Cecile Declais, Simon E. V. Phillips, David M. J. Lilley

    Research output: Contribution to journalArticle

    49 Citations (Scopus)

    Abstract

    T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 0.019 and 14 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.
    Original languageEnglish
    Pages (from-to)3505-3515
    Number of pages11
    JournalThe EMBO Journal
    Volume21
    Issue number13
    DOIs
    Publication statusPublished - 2002

    Keywords

    • Calorimetry
    • Crystal structure
    • DNA–protein interaction
    • Holliday junction resolvase
    • Nuclease

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