Deoxyribonucleases of Molecular Biology 15technique the DNA of interest is labeled at either the 3' or 5' end and
protein is bound to the DNA; the nucleoprotein complex is then sub-
ject to limited digestion with DNase I. Under the conditions used,
DNase will exhibit double-hit kinetics and, therefore, will insert ran-
dom single-strand nicks, however, cleavage will be inhibited if protein
is already bound to the DNA. The DNA is then purified, denatured,
and run on a sequencing polyacrylamide gel. Autoradiography of the
labeled DNA in the gel should show a ladder of fragments representing
cleavage at approximately every base for the control with no protein
present. However, if protein has been bound to a particular sequence,
there will be no cleavage by DNase I and there will be a gap in the
ladder. This gap is known as a footprint; the sequence of the footprint
can be determined if a sequencing reaction is run alongside the DNase
I protection assay. Detailed protocols for the technique are given in
refs. 14 and 15, also note that Stratagene now markets a DNA
footprinting kit using the DNase I protection method. Exonuclease III
protection has also been used for DNA footprinting, but in this tech-
nique the DNA must be labeled at the 5' end (see ref. 15).
3.8. Concluding Remarks
The DNases are a ubiquitous and diverse group of enzymes that play
an integral part in the functioning of DNA as the genetic material. Much
of the work on DNases has been directed toward finding useful tools for
molecular biology rather than toward elucidating their physiological
roles; despite this, only one of the DNases, DNase I, has found regular
use in techniques where limited digestion or complete digestion of
DNA is required. The prospects are that other eukaryotic DNase will
be found with more specific activities than DNase I, e.g., the enzymes
involved in DNA repair and DNases that may function in recombina-
tion events. Analysis and use of these enzymes will possibly give us
a greater insight into how DNA does function as the genetic material.References- Jones, M. and Boffey, S. A. (1984) Deoxyribonuclease activities of wheat seed-
lings. FEBS Lett. 174, 215-218.
2a. Melgar, E. and Goldthwaite, D. A. (1968) Deoxyribonucleic acid nucleases. 1.
The use of a new method to observe the kinetics of deoxyribonucleic acid degra-
dation by deoxyribonuclease I, deoxyribonuclease II and Escherichia coli endo-
nuclease I. J. Biol. Chem. 243, 4401-4408.