BAL 31 Nucleases 247
sites was analyzed in this laboratory for both the S and F nucleases, this
tendency was confirmed for the S enzyme and shown also to be true
for the F species (10). This suggests that the factors accounting for the
dependence of the rates of 3'-terminal nucleotide removal by the respec-
tive nuclease species on G + C content are not a direct result of the
relative thermodynamic stability of the two types of base pairs. This
is because the F nuclease, which removes 3'-terminal nucleotides more
rapidly and shows a lesser dependence of this rate on G + C content,
would otherwise be expected to show a lesser tendency to stop in
"runs" of several G-C pairs. Further support for the lack of a direct role
of relative thermodynamic stability of base pairs in this connection are
the facts that stops tend to occur at 5' dG residues, whereas stops at
both dC and dG residues should be expected if stability alone were the
dominant factor and the kinetic parameters show little dependence on
G + C content above about 50%, whereas the melting temperature Tm,
an indicator of stability, is linearly dependent on the G + C content.
Use of the F nuclease will make average extents of degradation more
predictable, however, because localized changes in velocity associ-
ated with local differences in G + C content are decreased.
- The Nuclease as a Probe for Lesions
in Nonsupercoiled Closed DNA
It was noted earlier that the nucleases can introduce breaks in double-
stranded DNA in response to a wide variety of both covalent and
noncovalent distortions of duplex structure, whereas covalently closed
circular duplexes that are essentially nonsupercoiled or slightly posi-
tively supercoiled (form I ° DNA) are extremely refractory to nuclease-
mediated attack (11,13,14). This provides for very stringent and general
tests of altered duplex structure caused by any perturbation that does
not introduce strand breaks (such breaks themselves provide substrate
sites) or result in significant negative supercoiling of form I ° DNA.
Information as to whether duplex DNA structure is perturbed by a
given reagent or treatment is valuable as a guide to further studies.
Form I ° DNA is readily produced from any supercoiled duplex
species by incubation with DNA topoisomerase I. This enzyme is
commercially available, but crude preparations (34) used as described
(11) are quite adequate for this purpose. The nicking-closing reaction
is routinely monitored by electrophoresis in 1% agarose gels.
