Methods in Molecular Biology • 16 Enzymes of Molecular Biology

(Nancy Kaufman) #1
BAL 31 Nucleases 233

increasing enzyme concentration (9). In the absence ofDNA polymerase-
mediated repair, ligation of partially shortened duplexes under condi-
tions favoring the joining of fully base-paired ends is undetectable
when low concentrations of nuclease are used, but becomes quite
substantial (up to 50%) at nuclease concentrations that give rise to tails
of minimum average length (9). This strongly suggests that the 5'-3'
exonuclease activity, acting on 5'-terminated single-stranded tails that
are generated through attack by the 3'-5' exonuclease on duplex ends,
terminates at the junction between single-stranded and duplex struc-
tures to leave fully base-paired ends on a significant fraction of the
molecules in a partially digested population. The length reduction of
duplex RNA (15) presumably proceeds by a similar mechanism, but
has not been characterized. The BAL 31 nucleases are the most effi-
cacious enzymes known for the controlled length reduction of duplex
DNA, and are apparently the only enzymes that can mediate this reac-
tion for duplex RNA.
The covalent lesions in duplex DNA that have been shown to elicit
cleavage by BAL 31 nuclease, some of which have been examined
only in the case of the S form, include strand breaks, nitrous acid-
induced inter-strand crosslinks, UV irradiation-induced photoprod-
ucts, adducts from reaction with arylating and alkylating agents or
with Ag ÷ and Hg ÷÷ ions, and apurinic sites (4,10,13,14). Quasiduplex
DNA, in which there are extra nucleotides in one strand separated by
3 bp, was attacked by the BAL 31 nuclease, but not by the S 1 and
Neurospora crassa nucleases (16). Noncovalent alterations in duplex
structure that can cause endonucleolytic attack include those associ-
ated with a very moderate degree of negative supercoiling and very high
positive supercoiling (12), as well as the junctions between regions of
B- and Z-DNA helical structures in the same molecule (8).
The only known nonsubstrate nucleic acids are nonmodified, nonsuper-
coiled (or slightly positively supercoiled) circular duplex DNAs, which
are cleaved at such slow rates that they serve as excellent controls for
the detection of lesions or distorted structures in such DNAs (14). The
strand break (nick) introduced as the initial cleavage event itself pro-
vides a substrate site, so that the other strand is cleaved, usually after
the removal of several nucleotides from the originally nicked strand at
the site of the nick (17), to produce termini that are then attacked so as
to shorten the resulting duplex as previously outlined.

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