Nucleases--An Overview 5
1.1.1.4. CLEAVAGE OF NATIVE OR SINGLE-STRANDED DNA
Nucleascs tcnd to havc a"prcfcrcnce" for cleavage of single-stranded
DNA or double-stranded DNA substratcs. In general single-strand
specific DNascs, such as nuclcasc SI fromAspergillus (EC 3.1.30.1),
will digest native DNA if the enzyme concentration is high. Nucleasc
S1 is used to analyzc the structure of DNA-RNA hybrids and in cDNA
synthesis whcrc it opens the hairpin loop gcncratcd during the synthesis.
1.1.1.5. GENERAL
DNascs have also in the past bccn classified according to their pH
optima and rcquircmcnts for thc presence of mctal ions; there arc two
major mammalian DNascs--onc working at neutral pH the othcr in
acidic conditions--and both rcquirc Mg ions. Other nuclcasc cnzymcs
exist that require Ca 2+ ions (pea nuclear DNasc [3]) or Zn 2+ ions
(nuclcasc S 1). In addition to the aforementioned propertics, it is important
to note that many crudc preparations of DNasc exhibit nonspccificity
for the sugar moiety of nucleic acids, i.e., they will cleave both RNA
and DNA. In these cases it is obviously essential to remove contami-
nating RNasc activity before the enzyme is used to remove DNA from
a preparation of RNA.
From the forcgoing discussion, it can bc sccn that the nuclcascs arc
a complex group of cnzymcs. However, when it comes to their use as
tools in molecular biology, the situation is very much simplified as
only a handful of enzymes arc used routincly in experimental proto-
cols. The enzymes DNascs I and II, cxonucleascs, nuclcasc S l, Bal3 l,
and RNasc will bc discusscd in much more dctail in the following
sections with special emphasis on the experimental protocols for thcir
use in molecular biology techniques.
References- Roca, J. and Mczquita, C. (1989) DNA topoisomcrasc II activity in nonrcpli-
caring, transcriptionally inactive, chickcn late spcrmatids. EMBO J. 8, 1855-1860. - Schmidt, G. and Laskowski, St., M. (1969) Phosphate ester cleavage, in Enzymes,
vol. 5b (Walkcr, P. D., Lardy, H., and Myrback, A., cds.), Acadcmic, Ncw
York, pp. 3-35.
3, Weir, A. F. and Bryant, J. A. (1989) Partial purification and properties of a
chromatin-bound dcoxyribonuclcasc from the embryo axes of germinating pea.
Phytochemistry 28, 1339-1343. - Bernardi, G. and Sadron, C. (1964) Studies on acid dcoxyribonuclcasc. I. Kinc-
tics of the initial degradation of DNA by acid DNase. Biochemistry 3, 1411-1418.