Ligases 217
range 0.2-1.0 mM cause a slight increase in activity, though higher con-
centrations are inhibitory (33). Co 2÷ and Ni 2÷ are inactive, Zn a÷ has slight
activity, and Ca 2÷ has been reported to yield 60% activity in some cases
and none in others (27,33).
T4 DNA ligase has a magnesium optimum of 10 mM, whereas Mn 2÷
is only 25% as efficient (17). However, in joining DNA:RNA hybrids,
Mn 2÷ is twice as effective as Mg 2÷ (34).
2.2.6. Activators and Inhibitors
Ammonium ions in low concentrations stimulate the E. coli enzyme, in
joining DNA termini, and Vma x can be increased by up to 20-fold (23). K ÷
and Rb ÷ show similar stimulation, Cs ÷ and Li ÷ exert a small effect, and
Na ÷ is ineffective.
In contrast, the T4 DNA ligase is unaffected by low concentrations
of ammonium ions, whereas higher levels (0.2M) of NH4 +, Na ÷, K ÷,
Cs ÷, and Li ÷ inhibit almost completely. Blunt-end ligation is inhibited
by 25 mM phosphate and 50 mM Na ÷ (35).
Polyamines, such as spermine and spermidine, also inhibit, but this
can be overcome by increasing the DNA concentration. Spermine is
the most effective, inhibiting the joining reaction by 90%. Some work-
ers, however, recommend the use of spermidine (35,36).
2.2.7. Sulfhydryl Reagents
E. coli DNA ligase does not need the presence of sulfhydryl reagents
(37). T4, T7, and mammalian DNA ligases do require either ]]-mercapto-
ethanol or dithiothreitol (DTT) (38). T4 DNA ligase performs much more
efficiently if DTT is the reagent of choice.
2.2.8. Enzyme Assay
There are several types of assays carried out to define the activity
of DNA ligase. Functional assays include the ligating of radiolabeled
DNA to unlabeled DNA in solution (33) or bound to a matrix (39), and
the conversion of poly A:T to a form resistant to exonuclease III (35).
Direct measurements can be made of the conversion of radiolabeled
phosphate monoesters to diesters resistant to alkaline phosphatase
activity (40). Also, the restoration of biological activity to DNA pre-
viously nicked by pancreatic DNAse or restriction endonucleases can
be used as an assay (3).