Biomedical Engineering Reference
In-Depth Information
responsible for this degradation is called a
restriction
endonuclease
or restriction enzyme (Lederberg &
Meselson 1964). The restrictive host must, of course,
protect its own DNA from the potentially lethal
effects of the restriction endonuclease and so its DNA
must be appropriately modified. Modification involves
methylation of certain bases at a very limited num-
ber of sequences within DNA, which constitute the
recognition sequences for the restriction endonucle-
ase. This explains why phage that survive one cycle
of growth upon the restrictive host can subsequently
reinfect that host efficiently; their DNA has been
replicated in the presence of the modifying methy-
lase and so it, like the host DNA, becomes methy-
lated and protected from the restriction system.
Although phage infection has been chosen as our
example to illustrate restriction and modification,
these processes can occur whenever DNA is trans-
ferred from one bacterial strain to another.
E. coli
K
EOP = 10
-4
λ
.K
EOP = 1
EOP = 1
EOP = 1
λ
.C
E. coli
C
Fig. 3.1
Host-controlled restriction and modification of phage
λ
in
E. coli
strain K, analysed by efficiency of plating (EOP).
Phage propagated by growth on strains K or C (i.e.
λ.
K or
λ.
C)
have EOPs on the two strains, as indicated by arrows.
Types of restriction and modification
(R-M) system
particular host strain depends upon the strain on
which it was last propagated. This non-heritable
change conferred upon the phage by the second
host strain (
E. coli
K) that allows it to be replated
on that strain without further restriction is called
modification
.
The restricted phages adsorb to restrictive hosts
and inject their DNA normally. When the phage
are labelled with
32
P, it is apparent that their DNA
is degraded soon after injection (Dussoix & Arber
1962) and the endonuclease that is primarily
At least four different kinds of R-M system are
known: type I, type II, type III and type IIs. The
essential differences between them are summarized
in Table 3.1.
The type I systems were the first to be character-
ized and a typical example is that from
E. coli
K12.
The active enzyme consists of two restriction sub-
units, two modification (methylation) subunits and
one recognition subunit. These subunits are the
Table 3.1
Characteristics of the different types of endonucleases.
System
Key features
Type I
One enzyme with different subunits for recognition, cleavage and methylation. Recognizes and methylates a single
sequence but cleaves DNA up to 1000bp away
Type II
Two different enzymes which both recognize the same target sequence, which is symmetrical. The two enzymes either
cleave or modify the recognition sequence
Type III
One enzyme with two different subunits, one for recognition and modification and one for cleavage. Recognizes and
methylates same sequence but cleaves 24-26bp away
Type IIs
Two different enzymes but recognition sequence is asymmetric. Cleavage occurs on one side of recognition sequence
up to 20bp away
