Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Deoxyribonucleases of Molecular Biology

A

9

3'

5'

5' 5'P. 5*P 5°P 3'

3' 5'P 5'

5' 5'P 5'P 5'P 3'

3' 5'P 5'P 5'P ~5'P .5'

B 5, .3,

3, .5,

5 ~ 3*P 3'P .3~P 3'

3' 3'P 3'P 3~P 5*

Fig. 1. A. Double-hit mechanism of DNase I in presence of Mg 2+ ions. B. Single-

hit mechanism of DNase II.

Apart from the monovalent cations, there is no general inhibitor of
DNase I such as those available for the inhibition of RNases. The only
real way to combat DNase activity, which may be a worry during DNA
extractions, is to do the extraction as quickly as possible and at low
temperatures. Inclusion of EDTA in the extraction buffer is a good
idea, but note that Jones and Boffey (1) recently discovered a DNase
in the leaves of wheat seedlings that appears to be stimulated in the
presence of EDTA.

2.4. Kinetics
Using a variety of methods, including light-scattering, viscometry,
and sedimentation analysis, it can be shown that there are two different
types of mechanism for the cleavage of ds DNA substrates by DNase
(Fig. 1). DNase I, at low concentrations and under the usual assay
conditions, inserts nicks at random points in each strand of the DNA
at points away from each other. This is termed a "double-hit" mechanism
(Fig. 1 A); complete scission of the molecule will not occur until two