234 Gray and Lu1.4. Uses in Manipulation of Nucleic AcidsThe duplex exonuclease activity of the nucleases has been very
widely exploited, because the removal of sequences from DNA ter-
mini in a controlled manner is desirable in numerous applications.
Unidirectional deletions can be done for DNAs cloned into a circular
vector if single unique sites for two restriction enzymes are present,
one on each side of the cloned insert. Linearization by cleavage with
one of the restriction enzymes allows for unidirectional deletions in
the insert, whereas subsequent cleavage with the other restriction enzyme
releases the partially degraded vector DNA so that the shortened insert
can be religated to an intact vector DNA for subcloning. Conversely,
manipulations of the vector DNAs themselves in this manner have
proven useful--an early application of this method led to a vector
DNA modified so that cloned inserts abutted the DNA coding for a
signal peptide of the vector, allowing secretion of the protein products
of cloned genes into the periplasmic space of E. coli (18,19). Such
unidirectional deletions in cloned inserts have been used as a means to
sequence, using a single primer for the DNA polymerase-mediated
extension, long segments of DNA cloned into M13 phage-derived
vectors through the production of a set of progressively shorter
unidirectionally deleted derivatives of the original insert (20). The
progressive removal of sequences from duplex termini allows the deter-
mination of the restriction map of a given DNA by observing the order
in which the fragments from digestion with the restriction enzyme in
question disappear from gel electrophoretic patterns of progressively
shortened samples (21). This technique is greatly enhanced if the frag-
ment to be mapped is in a circular cloning vector as ambiguity result-
ing from simultaneous degradation from both ends of the fragment is
eliminated (22). Cloning vectors containing infrequently cleaved restric-
tion sites have been constructed in this laboratory to take advantage of
BAL 31 nuclease-mediated deletions in both sequencing and restric-
tion fragment mapping (22).
The blockage of the exonuclease action by nucleosomes and by
interstrand crosslinks, such as can be induced by psoralen derivatives,
has been used to map the locations of such structures on duplex DNAs
(23,24). The detection of lesions or distortions in duplex DNA through
the endonuclease activity has been alluded to earlier.