Methods in Molecular Biology • 16 Enzymes of Molecular Biology

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BAL 31 Nucleases 243


corresponding to enzyme nonspecifically bound to duplex DNA away
from the ends and not catalytically active, and another productive
complex with enzyme bound at the termini, are needed to model this
reaction (10).
The significance of v0/[S] is that it is the number of nucleotide
residues released per DNA terminus per unit time (6), which is usually
the desirable parameter. This can be taken as equal to one-half the
number of base pairs removed per terminus for digests of more than
approx 50-100 nucleotides removed per terminus at enzyme concen-
trations that minimize single-stranded tail length (see Section 3.3.2.).
At very low nuclease concentrations, substantial single-stranded tails
may be present (9) so that the number of residues removed is not equal
to one-half the number of base pairs removed.
Data obtained in recent kinetic studies of the duplex exonuclease
activity to determine the effects of the G + C content of the substrate
for the S nuclease gave very large standard deviations for the values
of Kin app and Vm arp so that substitution of these values in Eq. (2) would
not give reliable estimates for v0/[S] (10). The reason proved to be that
the values of Kin app tend to be much larger than the largest values of [S]
compatible with the photometric technique, even after cleavage of the
DNA with a restriction enzyme to increase the number of termini per
unit weight concentration of DNA. Hence, the substrate concentration
range falls far short of the range of approx 0.33-2 gm app that is consid-
ered to yield optimally accurate results for such determinations (33).
In agreement with this, the plots ofv 0 vs [S] were all very good straight
lines, as expected from Eq. (2) where [S] << gmapp'~ the right-hand side
of Eq. (2) then reduces to VmapP/Km app, which will be constant at a fixed
enzyme concentration and is the slope of a plot of v 0 vs [S].
Hence, it is possible, in the range of [S] that will ordinarily be used
in such experiments, to calculate v0/[S ] at any given enzyme concen-
tration and data are moreover available to include the dependence on
G + C content as described in Section 3.3.3. As an example, consider
a test DNA of G + C content near 50 mol percent containing 5000 bp
that has been cut into six fragments by a restriction endonuclease, and
2 lag are treated with BAL 31 nuclease in a vol of 100 IlL. This would
be convenient for removal of several aliquots containing approx 0.5 lag
of DNA each for analysis (after quenching the reaction with an excess
of EDTA) at different times of exposure (i.e., 5-min intervals) to nuclease

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