124 Pingoud, Alves, and Geigerto p-mercuribenzoate, N-ethyl maleimide, 5,5'-dithiobis (2-nitro-
benzoic acid), and iodoacetate, whereas HindlII, BamHI, PvuI, AvaI,
SmaI, and PstI could be inhibited by some, but not all of the tested
sulfhydryl reagents.
The restriction enzyme catalyzed cleavage of DNA can be inhibited
by ligands that specifically bind to DNA such as certain antibiotics,
(distamycinA, netropsin, actinomycinD, anthramycin, olivomycin),
carcinogens (6,4'-diamidino-2-phenylindole), cytostatic drugs (cis-
dichlorodiamineplatinumlI), and dyes (proflavine, ethidium bromide,
Hoechst 33258) (74,160-171). This inhibition is dependent on the
recognition sequence and its flanking region, which reflects the pref-
erential binding of these drugs to AT- or GC-rich regions. It is, there-
fore, possible in some cases to suppress DNA cleavage by a restriction
enzyme at one site without affecting the cleavage at another site. The
effective concentration for inhibition may be different for single-strand
("nicking") and double-strand cleavage (171), an effect that has been
taken advantage of to study the kinetics of DNA cleavage (81,172) and
may be used to prepare open circular DNA with a specific
phosphodiester bond cleaved. Sequence-specific binding of oligo-
pyrimidine nucleotides to double-stranded DNA, which results in triple
helix formation, can be used, as was shown for Ksp632I (173) and
EcoRI (174), to inhibit restriction endonuclease catalyzed cleavage of
DNA at particular sites.
- Practical Considerations
for the Use of Restriction Enzymes
3.1. Storage of Restriction Enzymes
The stability of preparations of purified restriction enzymes criti-
cally depends on adequate storage conditions, in particular, on buffer
composition, enzyme concentration, presence of additives, and an appro-
priate storage temperature.
Different buffer compositions are recommended for the storage of
different enzymes: For most restriction enzymes 10-50 mM Tris-HC1,
pH 7.5, a minimum of 50 mM NaC1 or KC1, 1 mM EDTA, and 60%
(v/v) glycerol have proven to be optimal. Many restriction enzymes
require additional additives, most commonly 1 mM 1,4-dithiothreitol,
1,4-dithioerythritol or [3-mercaptoethanol (to prevent oxidation of