246 Gray and Luwith S 1 nuclease or DNA polymerase to remove single-stranded ends
and provide fully based-paired termini is unnecessary, although it is
apparent (9) that polymerase-mediated repair will enhance the frac-
tion of ligatable termini.
3.3.3. Effects of Base Composition
The duplex exonuclease activity of the S form decreases markedly
with increasing G + C content (8, 21), and the magnitude of this effect
ha~been examined over the range of 37-66 mol% G + C residues (10).
For the S nuclease, the effect is significant and leads to an approx 4.2-
fold change in v0/[S ] over this composition range. The dependence of
v0/[S] on G + C content over the above range can be approximated,
independently of the substrate concentration as previously noted, by
the equation:
v0/[S] = 16.85 - 0.546P + 0.00456P 2 (3)where P is the mol% G + C, and the value of v0/[S] is that correspond-
ing to an enzyme concentration of 1 U/mL. The desired value of v0/[S]
is thus calculated by multiplication of that from Eq. (3) by the nuclease
concentration (U/mL) to be used in the actual experiment. The equa-
tion should not be used to extrapolate outside of the above range of
base composition (37 _< P < 66).
The duplex exonuclease activity of the F enzyme is much less depen-
dent on composition than for the S enzyme, with Vo/[S] varying only
by a factor of 1.7 over the above range at the substrate concentration
of the example used earlier. Moreover, nearly all the change occurs
between approx 52 and 37% G + C. Given the paucity of data, the
values for the maximum velocity/U/L of nuclease and Km app above
should be used in the range of 65---52% G + C, and the maximum velocity
parameter should be increased by interpolation from the value above
to a maximum of 1.7 times that value in the range from 52-37% G + C.
The less pronounced effect of variances of G + C content for the F
nuclease suggests its use where it is desired to minimize the effects of
local sequence features, such as local regions of high G + C content,
on the termination points of digestion of partially degraded samples.
With samples that corresponded to S nuclease, sequence analysis of
partially degraded samples showed a strong tendency for the nuclease
to stop in regions of several consecutive G + C pairs with a strong
preference for dG at the 5' end (18,19). When the location of "stop"