Restriction Enzymes 123or d(TCTCCGGTT) by MspI (153) may be owing to duplex formation
via the palindromic -CCGG- core sequence, similarly described for
the cleavage of "single-stranded" oligodeoxynucleotides by EcoRI
(106). In a more recent report, Bischofsberger et al. (154) concluded
from cleavage experiments carried out with immobilized oligodeoxy-
nudeotides that EcoRI is capable of cleaving single strands. Since it
cannot be completely ruled out that even oligodeoxynucleotides cova-
lently linked to oligo-dT-cellulose can in part form duplex molecules
via the palindromic recognition site, this conclusion should be met
with reservation. Similarly, the reported cleavage of DNA in DNA x
RNA hybrid double strands by EcoRI, HindlI, SalI, MspI, HhaI, AluI,
TaqI, and HaelI (155) presumably can be explained by normal DNA
cleavage, since the substrate was produced by the reverse transcrip-
tion of a viral RNA by AMV polymerase, was not characterized with
respect to a DNA x RNA hybrid structure and was not analyzed as to
what extent it was contaminated with normal DNA double strands,
which are also produced by AMV polymerase.
2.5. Inhibition
The cleavage of DNA by restriction enzymes can be inhibited by
covalent modification of the substrate or the enzyme, as well as by
complex formation with low-mol-wt ligands. Chemical modification
of restriction enzymes has been used to characterize their active sites:
BglI catalyzed cleavage of DNA is inhibited by 2,3 butane-dione modi-
fication of Arg residues, DNA binding remaining unaffected (53). For
Eco RI, it was shown that the modification of Lys residues with methyl
acetimidate inactivates the enzyme; the relevant Lys residues are pro-
tected against chemical modification by complex formation with the
substrate (67). It was also shown that Glu residues are essential for
catalysis of EcoRI, since modification with 1-cyclohexyl-3-(2-mor-
pholinoethyl) carbodiimide metho-p-sulfonate inactivates the enzyme
(156). Rosebengal-sensitized photooxidation of His residues leads to
the inactivation of EcoRI, but not EcoRV (157). In contrast, although
modification of a single sulfhydryl group by a fluorescent maleimide
derivative leads to the total loss of activity in EcoRV, it does not affect
the activity of EcoRI (158). In a detailed study, Nath (159) has ana-
lyzed the effect of several sulfhydryl reagents on restriction enzyme
activities: SalI, BgllI, MpaI, EcoRI, and SstlI proved to be insensitive