236 Gray and Lupairs. Moreover, the half-life of the activity on single-stranded DNA
and the duplex exonuclease activity are 3-5 min at 50°C (10). The
half-life of the activity is at least 20 h at 30°C, which is the recom-
mended temperature for manipulations with these enzymes.
In a storage buffer containing 5 mM each of Ca 2÷ and Mg 2÷ near
4°C, the nuclease should retain most of its activity on a time scale of
years as inferred from studies in which no activity was lost upon stor-
age for several months (4). Most suppliers provide the enzyme in 50%
(v/v) glycerol for storage at -20°C, in which it should maintain full
activity indefinitely.
2.4. Effects of Ionic Strength,
Protein Denaturing Agents,
and Other Potential Inhibitors
A concentration of NaC1 of 0.6Mis present in the usual assay buffer.
The activity on single-stranded DNA is optimal in this range (4), but
the duplex exonuclease activity appears to increase if the ionic strength
is reduced from that of the assay buffer (26). However, the NaC1 con-
centration of the usual buffer will repress the activity of contaminating
salt-sensitive nucleases or other factors that can cause adventitious breaks.
The nuclease was used at 0.1M NaC1 with no evidence of internal
breaks in nonsupercoiled closed duplex DNA provided that the buffers
were autoclaved; omission of this step did lead to the appearance of
unexpected strand breaks (12). Such internal breaks must be avoided
because each such lesion, after cleavage of the intact strand, gives rise
to two new termini, which then serve as substrates for the duplex
exonuclease activity.
Very high concentrations of 1:1 electrolytes are tolerated extremely
well by the nucleases. With single-stranded DNA as substrate, the S
enzyme displays 40 and 27% of its maximal activity in 4.5M NaCI and
7M CsC1, respectively (4); in a comparison at only two concentrations,
the F nuclease was 70% as active in 4.5M NaC1 as in 0.6M NaC1 (8).
The duplex exonuclease activity of the F nuclease at 4.5M NaC1 was
26% of that at 0.6M (8).
Protein denaturing agents at concentrations that normally denature
proteins are unable to abolish the BAL 31 nuclease activities. Single-
stranded DNA was hydrolyzed at 40% of the maximal rate in 6.5M
urea (4), whereas crude preparations maintained activity against both