118 Pingoud, Alves, and Geigersequences adjacent to the recognition site have shown for EcoRI that
AT base pairs enhance and GC base pairs slow down the hydrolytic
activity of EcoRI, presumably because the structure of the recognition
sequence or its propensity to adopt a certain conformation is modu-
lated by the flanking sequences such that it becomes a better or worse
substrate for EcoRI (106). Similar mechanisms are likely to operate
with all restriction enzymes (107).
2.3.3. Order of Reaction
In Fig. l, the DNA cleavage reaction catalyzed by a restriction
enzyme is formulated as two partial reactions that lead to the hydroly-
sis of a phosphodiester bond in each of the two individual strands of
the DNA duplex. Depending on the DNA substrate and the reaction
conditions, these two partial reactions can be separated kinetically,
when the rate of cleavage of the first phosphodiester bond is smaller
than the rate of dissociation of the enzyme from the nicked substrate,
or the two partial reactions take place in a concerted fashion, when the
rate of cleavage of the second phosphodiester bond is higher than the
rate of dissociation (75,108-111). In general, restriction enzymes fol-
low a concerted reaction mechanism under optimum reaction condi-
tions, unless the base composition of the sequences flanking the
recognition site is very different. In these cases, the rates of cleavage
of the first and second strand can become very different, such that the
enzyme has an increased probability to dissociate from the nicked
intermediate. DNA substrates in which the symmetrical recognition
site for a restriction enzyme has been made unsymmetrical by intro-
ducing phosphorothioate groups in one strand (112,113) or a single
mismatch base pair (114) are cleaved much better in the unmodified
than in the modified strand, which allows the isolation of a nicked
intermediate. The preferential attack of the unmodified strand in double-
stranded DNA containing phosphorothioates by restriction enzymes
has been taken advantage of to remove the template single strand by
exonuclease digestion for in vitro mutagenesis experiments.
Phosphorothioate substituted oligodeoxynucleotides have been used
to elucidate the stereochemical course of the EcoRI catalyzed cleav-
age reaction: Hydrolysis of the Rp diastereomer of d(pGGsAATTCC)
proceeds with inversion of configuration at phosphorous, suggesting
that hydrolysis occurs by a direct nucleophilic attack of water at the