Mung-Bean Nuclease I 257
The relative base preference of mung-bean nuclease is A > T(U) >
C > G as reported by Mikulski and Laskowski (6), who noted that the
ribohomopolymer poly-U was preferentially degraded with respect to
poly-A. This is thought to be due to the fact that poly-U lacks a rela-
tively ordered secondary structure. The structure of the DNA at A,T-
rich regions has been implicated in the apparent preference of the
nuclease for these regions. A good example of this is phage ~, DNA,
which is preferentially cleaved in the A,T-rich central portion of the
molecule (3). Other sites are cleaved more slowly. Johnson and
Laskowski (3) suggest that regions of DNA rich in A,T residues pos-
sess a certain degree of single-stranded character (dependent upon
region length, temperature, and ionic strength). This suggestion is
based on the work of von Hippel and Felsenfeld (14) and Wingert and
von Hippel (15), who postulate that, at regions below T m A,T-rich
regions undergo local strand separation to a greater extent than G,C-
rich regions, a phenomenon known as "structural breathing." A more
recent explanation is that "stable DNA unwinding," not "breathing"
accounts for mung-bean nuclease 1 hypersensitivity of specific A,T-rich
regions (16). These regions of stable DNA unwinding are of potential
biological importance, since they have been detected in replication
origins and transcriptional regulatory regions. Kowalski et al. (16)
found that nuclease hypersensitivity of the A,T-rich regions is hierar-
chical, and either deletion of the primary site or a sufficient increase
in the free energy of super-coiling leads to an enhanced nuclease cleav-
age at an alternative A,T-rich site.
Vernick et al. (17) have suggested the involvement of an altered DNA
structure near gene boundaries in determining the recognition sites for
this enzyme, since they found no direct relationship between dA-dT-
richness and site of cleavage. As yet, no specific recognition sequence
has been determined for mung-bean nuclease 1, although there is some
evidence to suggest that the enzyme may be "region-specific" (3).
2.8. Stability
Pure mung-bean nuclease is stable at pH 7-8 in buffer, although this
is not its optimum pH for enzyme activity. However, Kowalski et al.
(9) reported that the enzyme can be stabilized at pH 5.0 in the presence
of 0.1 mMZn acetate, 1.0 mM cysteine, and 0.001% Triton X-100. It
is stable to heat treatments up to 65°C (60-70°C). The purified enzyme,