256 Sharp and Slater2.7. Specificity
Mung-bean nuclease 1 has more than one activity. Its predominant
mode of action, and that for which it is most commonly used, is as a
single-strand-specific, but sugar nonspecific nuclease that will cleave
the 3'-phosphodiester bonds of a single-stranded substrate, giving rise
to mono-, di-, and trinucleotides.
Mung-bean nuclease 1 will degrade double-stranded DNA under
certain conditions, i.e., under conditions that do not favor a tight helical
DNA structure, large duplex polymers of DNA are completely degraded
from their termini. The terminally directed single-strand- and double-
strand-specific activities of the enzyme are both intrinsic properties of the
molecule, the evidence being that (1) they are inactivated and reactivated
in parallel and (2) the two activities migrate together on analytical gels (4).
Under conditions that favor the tight helical structure of DNA, the
enzyme has been shown to catalyze a number of double-stranded cleav-
ages at a limited number of points. This occurs by a two-step mecha-
nism: first, the creation of a single-stranded nick, then a double-stranded
scission (4). Other workers have shown that the sensitivity of duplex
DNA toward endonucleolytic scission of one strand increases with
increasing superhelical density. This is thought to result from tight
bends in the DNA. Such distorted regions become favored sites for
single-strand-specific nucleases. The degree of super-coiling is impor-
tant with respect to nuclease cleavage (12). In addition to terminally
directed single-stranded and double-stranded nuclease activity and
the limited endonucleolytic activity on duplex DNA under the correct
conditions, there is another separate activity that appears to reside on
the same molecule and copurifies with it. Mikulski and Laskowski (6)
reported a Y-o~-monophosphatase activity that cleaved the 3'-terminal
phosphate group of 3'-mononucleotides and that was distinct from the
Y-nuclease activity (i.e., that which cleaves the phosphodiester bond
between nucleotides of a polynucleotide chain). Both of these activi-
ties will result in an --OH group at the 3' position. This monophos-
phatase activity hydrolyzes ribose mononucleotides 50-100 times faster
than it hydrolyzes deoxyribose mononucleotides. This example of
activity toward both phosphomonoester and phosphodiester bonds
residing in the same enzyme is not unique. Richardson et al. (13) reported
similar activity with exonuclease 111.