186 Pingoud, Alves, and Geigerrecognition model, the new one is highly redundant, a result that had
emerged already from previous site-directed mutagenesis experiments
on EcoRI (reviewed in ref. 201). The revised structure of the EcoRI-
DNA complex is in agreement with previous (201) and more recent
(202) mutational analyses.
Although the structure of the EcoRV-DNA complex has not yet
been published, its" general features have been reviewed (203,204).
The DNA is highly deformed in the complex. Recognition interactions
comprise 12 protein-base hydrogen bonds and several van der Waal's
contacts, as well as electrostatic contacts. Results from site-directed-
mutagenesis experiments support the recognition model derived from
the X-ray structure analysis (205-207). Different from EcoRI, EcoRV
does not bind to DNA in a specific manner when Mg 2÷ is absent (208);
it does so, however, in the presence of Mg 2÷ (209). EcoRV does not
seem to be an exception, because, as with TaqI, specificity is observed
only late in the enzymatic cycle (210). The structural reason for the
different behavior of EcoRI and EcoRV--as the paradigmatic examples
of restriction enzymes that exhibit DNA binding specificity already in
the absence or only in the presence of Mg2÷---could be the degree of
overlap of the DNA binding site and the catalytic center. In EcoRI,
which produces a four-base extension, most of the specificity deter-
mining contacts can presumably be formed in the absence of Mg 2÷,
because electrostatic repulsion between DNA and protein owing to
several negatively charged amino acid residues of the Mg 2÷ binding
site occurs only at the end of the recognition sequence. In EcoRV,
which is a blunt-end cutter, negatively charged amino acid residues of
the Mg 2÷ binding site are located vis-h-vis the center of the recognition
sequence in the protein-DNA complex; specificity determining con-
tacts, therefore, can hardly be formed in the absence of Mg 2÷.
Structural and mechanistic studies on EcoRI and EcoRV have not
only made it possible to identify amino acids that are involved in
specific DNA binding, but also those that are likely to have a primarily
catalytic function (205,206). Based on this information, and knowing
that EcoRI and EcoRV cleave the phosphodiester bond with inversion
of configuration (115, 211), a catalytic mechanism, which is sufficiently
general to apply for many restriction enzymes (212), was proposed for
these enzymes. Important features of this mechanism include water
activation by a phosphate residue next to the scissile phosphodiester