108 Pingoud, Alves, and GeigerTable 1
Classification of Restriction Enzymes
Type I Type II Type III
Example EcoK EcoRI EcoP
Subunits Three different Two identical Two different
Activity Restriction, modi- Only restriction Restriction, modifi-
fication, topo- cation, ATPase
isomerase, ATPase
Cofactor Mg 2÷, ATP, Mg 2÷ Mg 2÷, ATP,
requirements S-AdoMet (S-AdoMet)
Recognition AACNNNNGTGC GAATTC AGACC
sequence
Position of Variable and great Within the 25 bp away from
cleavage distance from the recognition the recogni-
recognition site site tion sitemodification enzyme, which was supposed to recognize the same spe-
cific DNA sequence on the host DNA, methylate it, and thereby pre-
vent cleavage. This was verified in vitro by Meselson and Yuan (3),
who demonstrated that bacteriophage ~, DNA isolated from an E. coli
C culture was degraded by the E. coli K restriction enzyme, whereas
bacteriophage ~, DNA isolated from an E. coli K culture was not. A
similar experiment was carried out by Linn and Arber (4) with bacte-
riophage fd DNA. Later Smith and Wilcox (5) showed that the
Haemophilus influenzae restriction enzyme cleaved DNA from the
bacteriophage P22, but had no effect on the chromosomal DNA.
On detailed biochemical characterization of purified restriction
enzymes (3-6), it became apparent that they differed in their basic
enzymology, in particular their subunit composition, cofactor require-
ment, and mode of cleavage. Three classes are recognized: Type I (EC
3.1.21.3), Type II (EC 3.1.21.4), and Type III (EC 3.1.21.5) (Table 1).
Type I enzymes, such as EcoK (3), typically are composed of three
nonidentical subunits, need Mg 2÷ ions, ATP, and S-adenosylmethionine
to be active, and cleave the DNA at apparently random sites far away
from the recognition site. Type II enzymes, such as HindlI (5), typi-
cally exist as dimers of two identical subunits, require only Mg 2÷ ions
for their activity, and cleave DNA within or very close to the recogni-