ESR spectroscopy identifies inhibitory Cu2+ sites in a DNA-modifying enzyme to reveal determinants of catalytic specificity.

ABSTRACT: The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu(2+) ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu(2+) coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu(2+)-ion based long-range distance constraints. Double electron-electron resonance data yield Cu(2+)-Cu(2+) and Cu(2+)-nitroxide distances that are uniquely consistent with one Cu(2+) bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu(2+) ions bind per complex. Unexpectedly, Mg(2+)-catalyzed DNA cleavage

SUBMITTER: Yang Z 

PROVIDER: S-EPMC3340070 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature