122 Pingoud, Alves, and Geigerconditions of elevated pH and low ionic strength, EcoRI cleaves DNA
not only within the canonical recognition sequence -GAATTC-, but
also at other sites. A variety of other conditions reduce the sequence
specificity of EcoRI, including the presence of Mn 2÷ instead of Mg 2÷
(59,134) and the presence of organic solvents, such as glycerol or
dimethyl sulfoxide (135-138). It was suggested that this "EcoRI*
activity" was directed against sites containing the sequence -AATT-.
Later, it was deduced by Gardner et al. (139) that under "star" condi-
tions EcoRI recognizes sites that differ in a single position from the
canonical EcoRI sequence; any substitution can occur, with the excep-
tion of A ---> T or T ---> A changes within the central tetranucleotide.
Several other restriction enzymes have been shown to exhibit "star"
activities, e.g., AvaI (140), BamHI (138,141), BstI (142), BsuI (143),
EcoRV (144), HaelII (140), HhaI (138), HindlII (134,145), HpaI (140),
PstI (138), PvulI (146), SalI (138,140), SstI (138,140), SstlI (140),
TaqI (147), and XbaI (138,140). For BamHI, EcoRI, EcoRV, HindlII,
PvulI, and TaqI the "star" site cleavage sequences have been deter-
mined to differ from the normal recognition sequence only by a
single base pair.
"Star" patterns have been observed in restriction digests also under
optimum buffer conditions when high concentrations of the restriction
enzymes were used; the kinetics of cleavage are different, however,
since suboptimal buffer conditions decrease the high accuracy of restric-
tion enzymes by lowering the rate of cleavage at canonical sites and
increasing the rate of cleavage at degenerate sites, whereas at high
concentrations of enzyme under optimum buffer conditions, cleavage
at canonical sites occurs with a very much higher rate than at degen-
erate sites. It has been shown that the presence of spermine and
spermidine in the reaction buffer effectively suppresses "star" activity
of BamHI, BsuRI, EcoRI, EcoRV, HindlII, PstI, and SalI (148).
2.4.3. Single-Strand Cleavage
Several restriction enzymes were reported to cleave single-stranded
DNA, for example, HaelII (149), HhaI and SfaI (150), and HaeIII, HhaI,
Hinfl, HpalI, and MbolI (151). Detailed mechanistic studies, however,
demonstrated that most likely "single-strand" cleavage occurs at tran-
siently formed double-stranded sites (152). This is presumably also true
for oligodeoxynucleotide substrates; the cleavage of d(GAACCGGAGA)