Deoxyribonucleases of Molecular Biology 11
promote the binding of two DNase I molecules at opposite sites on the
two DNA strands (it is known that active DNase II is dimeric); or (2)
the ability of Mn a÷ to enable the DNase I enzyme molecule to flip
from the hydrolyzed strand to the opposite strand, which is then also
cleaved (2b).
2.5. Specificity
There is evidence that certain deoxynucleotide sequences can be
hydrolyzed in the presence of Mn 2+ but not in the presence of Mg 2+.
For example, poly (dG:dC) can be hydrolyzed by DNase I with Mn 2÷
but not with Mg2÷(4). This resistance is not attributable to secondary
structure of the double-helix, as DNase I in the presence of Mg 2÷ is
not able to hydrolyze the dC strand of a polymer consisting of
dI:dC. Therefore, in addition to altering the kinetics of the reaction,
different metal ions can also place qualitative constraints on hydroly-
sis of certain residues.
- Experimental Procedures
3.1. Introduction
DNase I is the enzyme of choice for all molecular biology tech-
niques (apart from restriction digestions) that require a double-stranded
DNase enzyme. It is readily available in pure form, i.e., exonuclease
and RNase free, from a number of commercial sources, has high acti-
vity on ds DNA, and at high concentrations, will also cleave ss DNA.
The reaction does not go to completion, to produce deoxynucleotide
monomers, but a certain limit size is reached, after which no further
digestion takes place. DNase I cleavage leads to the formation of 5'
monoesterified products and the enzyme has optimum activity in the
pH 7.0 region. DNase I is inhibited by monovalent cation but has an
absolute requirement for divalent metal ions. The nature of the cation
affects the mode of action of the enzyme and its ability to cleave at
certain deoxynucleotide residues. The enzyme has optimal activity in
the presence of Mn 2+ cations, but it is noteworthy that molecular bio-
logists use Mg 2+ as the cofactor. This is probably attributable to the
fact that many of the enzymes used in conjunction with DNase also
require Mg 2+ as cofactor, e.g., DNA polymerase I in the nick-transla-
tion protocol, and that many of the techniques require only limited
digestion of the DNA.