4 Weir1.1.1. Criteria Used for Classification
1.1.1.1. Exo- vs ENDONUCLEOLYTIC CLEAVAGE
Exo-DNases cleave from the ends of DNA molecules releasing
phosphomononucleotides. Cleavage can be either in the 3' to 5' direc-
tion releasing 5' phosphomononucleotides or in the 5' to 3' direction to
yield 3' phosphomononucleotides. An example of a widely used exo-
nuclease is exonuclease III fromEscherichia coli (EC 3.1.11.2), which
will digest one strand of a double-stranded DNA molecule from a 3'
overhang or blunt end. This property has been used to produce bidirec-
tional or unidirectional nested deletion of templates for sequencing.
Endo-DNases cleave at internal phosphate bonds. Cleavage of
double-stranded DNA substrates can be by a "single-hit" or a "double-
hit" mechanism (4) or by a combination of both (see Chapter 2, Section
2.4.). Essentially this means that the enzymes can either cleave the two
strands of the DNA molecule at points opposite or at sites on the two
strands that are well away from each other. The scission of the mol-
ecule will take place at a relatively faster rate in the former case as
compared to the latter. The prime example of an endo-DNase is pan-
creatic DNase (DNase I, EC 3.1.21.1). Under optimal conditions this
enzyme uses a double-hit mechanism for cleavage of substrates.
1.1.1.2. BASE SPECIFICITY AT OR NEAR THE SITE OF CLEAVAGE
None of the eukaryotic enzymes so far isolated appear to have such
specificity, however there is evidence that enzymes with optimal activity
on single-stranded DNAs will preferentially cleave atA-T rich sites in
native DNA molecules (3,5,6). As already mentioned, the Type II
restriction endonucleases have absolute specificity for a group of bases
at or near the cleavage site.
1.1.1.3. SITE OF CLEAVAGE
The site of cleavage can be on either side of the phosphate bond
leading to a 5' or a 3' monoesterified product. No enzymes have been
isolated that can split the internucleotide bond on either side. This
property is particularly important if the DNA molecule is to be subse-
quently made blunt-ended for a ligation experiment. DNA molecules
leh with a 5' overhang from a staggered cut are usually filled in with
the Klenow fragment ofE. coli DNA polymerase, whereas those with
a 3' overhang have the overhang cleaved back with the exonuclease
activity of T4 DNA polymerase to create blunt ends.