BAL 31 Nucleases 235
- Enzyme Requirements
2.1. pH
The near-neutral pH optima for the nuclease-catalyzed reactions
(4,9,25) is an advantage for most work. There is a difference in the pH
optima for single-stranded and linear duplex substrates for both the S
and F nucleases, which are in the ranges 8.5-8.8 and 7.0-8.0 for single-
stranded and linear duplex DNA, respectively (4, 9,25). Attack on super-
coiled DNA, examined only for the S enzyme, was optimal at the same
pH as for linear duplex DNA (4).
2.2. Metal Ion Cofactors
Ca 2+ and Mg 2÷ are both required cofactors (3). Ca 2+ is essential for
activity, with nuclease activities on both single- and double-stranded
DNA irreversibly (with respect to the readdition of excess Ca 2÷) lost
in solution if the molar concentration of EDTA exceeds that of this
cation (3,4). However, activity can be recovered after electrophoresis
under denaturing conditions (sodium dodecyl sulfate [SDS]-polyacry-
lamide gels) by incubation in a Ca2+-containing buffer (7).
Maximum velocity against both single-stranded and linear duplex
DNAs at constant [Ca 2÷] is achieved between 10 and 15 mM Mg 2÷
(9,25). At nominally zero (actually 0.01-0.02 mM) Mg 2÷, there was
residual duplex exonuclease activity (8 and 45% for the S and F
enzymes, respectively), but virtually none for single-stranded DNA.
In corresponding profiles where [Ca 2÷] was varied, concentrations
near 10 mM are needed to achieve full velocity for the length reduction
of duplex DNA, but activity is maximal on single-stranded DNA at or
below 1 mM (9,25).
In light of the above, a buffer containing 12.5 mM each of Ca 2÷ and
Mg 2÷ is recommended, since this confers full activity with respect to
both classes of substrate. The 5-mM concentrations of these ions in the
BAL 31 nuclease buffers recommended by some suppliers would yield
only 60-65% of the duplex exonuclease activity.
2.3. Effects of Temperature
The optimal temperature for the activity on single-stranded DNA is
near 60°C (4), but the use of such elevated temperatures is impractical.
Internal breaks would almost certainly be introduced into duplex DNA
because of at least transient thermally mediated unstacking of base