BAL 31 Nucleases 245
nuclease/L and 58 + 2 nM for gm app. Vma app in Eq. (2) is obtained
simply by multiplying the normalized maximum velocity above by the
nuclease concentration in U/L (the velocity was shown to be linear
with enzyme concentration for both the F and S enzymes over a wide
range of substrate concentration) (9,25). The dependence of v0/[S ] on
%(G + C) is much weaker than for the S enzyme (see Section 3.3.3.).
Several comments are in order here. The DNA should be precipi-
tated and resuspended in a small volume of Tris/EDTA buffer, and
diluted into a reaction mixture that will give the desired buffer com-
position (same as that for the assay using single-stranded DNA), since
it will be difficult to resuspend directly in the reaction buffer. The
expected extent of degradation, at least in the first one or two aliquots,
should not be more than approx 20% of the average fragment length
so that smearing of the bands of asynchronously digested DNA will
not preclude reasonable molecular size estimation from at least some
of the fragments. To avoid changes in substrate concentration owing
to complete digestion of fragments, a restriction enzyme that produces
any very short fragments from the DNA should be avoided. Because
the velocity does decrease with time, apparently because of inhibition
by released 5'-dNMPs (6), it should not be assumed that extensive
digestion of a DNA (more than 25%, for example) will proceed at the
same rate as that observed early in the course of a digest.
3.3.2. Effect of Nuelease Concentration
The presence of substantial single-stranded tails on partially digested
duplexes can be avoided if the concentration of nuclease is near 2 and
10 U/mL for the F and S enzymes, respectively, or greater (9). The
value corresponding to S nuclease should be used for all commercial
preparations not sold as separated S and F enzymes. These (or higher)
concentrations are compatible with most uses of the enzymes, corre-
sponding to 33 and 8 residues removed/terminus/min from a DNA of
near 50% G + C content for the F and S nucleases, respectively, at the
substrate concentration (7.3 × 10-8M, needed only for the calculation
for F nuclease) of the above example. Significantly, digestion at or
above the aforementioned concentrations not only leaves short aver-
age tail lengths (approx 7 residues), but allows ligation under condi-
tions favoring blunt-end joining of up to 50% of that observed with
DNA known to possess fully base-paired ends (9). Hence, treatment