10 Weirnicks are opposite. Monitoring of the reaction therefore will not indicate
the presence of DNA molecules of intermediate size until after a lag
phase (2a). In contrast, DNase II was shown to cleave high-mol-wt
DNA substrates on both strands at points opposite to each other result-
ing in the complete scission of the molecule (3). This process was termed
a single-hit mechanism (Fig. 1B), as scission of the DNA occurred
from a single encounter with the enzyme molecule; during a digestion,
intermediate size molecules will appear immediately. An interesting
feature of DNase I is that the cleavage mechanism can be altered from
double-hit to a DNase II-like single-hit mechanism by using high concen-
trations of the enzyme or by altering the divalent cation from Mg 2÷ to
Mn 2÷ or Co 2÷. Using ss DNA as substrate, Melgar and Goldthwaite
(2b) showed that in the presence of Mn 2÷ ions, DNase I had vastly
increased Vma x as compared to the activity in the presence of Mg 2÷,
whereas there was little change in the K m. The increased rate of hydro-
lysis in the presence of Mn 2÷ could of itself lead to the formation of
intermediate size fragments in the short periods observed without there
being a change in the actual mechanism of cleavage of a double-stranded
substrate; the greater the number of random nicks, the greater the
likelihood of there being scission of the molecules. If viscosity measure-
ments are used to follow the progress of a DNase digestion, a relation-
ship can be obtained when the log of a function of the change in
viscosity of the DNA solution is plotted against log time. The slope of
the resulting line, n, gives an indication of the mechanism of the reac-
tion: A value of approx 1.0 indicates single-hit kinetics, whereas a value
between 1 and 2 indicates a predominantly double-hit mechanism. When
the hydrolysis ofds DNA by DNase I in the presence of Mn 2÷ is monitored
by viscometry at low temperature, i.e., at low rate of hydrolysis, a value
of n = 1.16 is obtained, indicating that the reaction is predominantly of
the single-hit kind. Monovalent cations also lower the rate of hydroly-
sis of ds DNA by DNase I in the presence of Mn 2÷, and under these
conditions the n value changes from approx 1.0 (single-hit mechanism)
to values approaching 2.0 (double-hit mechanism). The inhibition of
DNase I by monovalent cations is probably a result of competition for
effector sites that directly or indirectly affect the active site of the enzyme.
The process by which Mn a÷ promotes the hydrolysis of ds DNA by
DNase I to switch from a double-hit mechanism to a single-hit mecha-
nism is not known but may involve either (1) the ability of Mn 2÷ to