238 Gray and LuSystematic studies have been done on the effects of a "chaotropic"
agent (NaC104) and compounds that protect cells from damage due to
increases or decreases in extracellular osmolarity (10). However, these
are not compounds that are likely to be part of a reaction mixture in
general applications of these enzymes, and it is noted here only that
NaC104 rapidly inactivates the nucleases at concentrations in the
range of 1-2M.2.5. Effects of Proteolysis
The proteolytic conversion of F to S nuclease that takes place in
Alteromonas culture supernatants can be mimicked by proteolysis in
vitro (7). However, it has also been found that extended exposure to
protease of the S nuclease can preferentially remove the duplex exo-
nuclease activity, but the bulk of the activity against single-stranded
DNA survives. The ability to linearize a nicked circular DNA corre-
lates with the remaining activity on single-stranded DNA, so that the
endonuclease activity in response to lesions is not lost upon proteoly-
sis. Since the bulk of the activity on single-stranded DNA is of the
5'-3' exonuclease variety, whereas attack on duplex DNA is through
a 3'-5' mode, the data suggest that the protease-treated nuclease may
lose its 3-5' exonuclease capability (7). The observation is interesting,
because if conditions for the removal of the duplex exonuclease activ-
ity can be optimized, the remarkable power of the enzyme to cleave
endonucleolytically in response to alterations in duplex DNA struc-
ture could be much better exploited to reveal imperfections, such as
small mismatches in nominally duplex DNAs, since the information
as to the site of cleavage would not be obliterated by the exonuclease
activity.
The internal breaks that were thought to be introduced into the S
nuclease by proteolysis to produce the preferential loss of duplex
exonuclease activity were not revealed in denaturing polyacrylamide
gels, where the proteins are heated in boiling water in the presence of
1% SDS and 20 mM [3-mercaptoethanol prior to electrophoresis. Rather,
the mol mass appeared to be unaffected for samples that had clearly
undergone loss of over 90% of the starting duplex exonuclease activity,
but retained over half of the activity against single-stranded DNA. When
urea was added to the denaturation buffer to 6M, the internal breaks
were revealed by the progressively more extensive fragmentation of