Restriction Enzymes
Restriction Enzymes
Restriction Enzymes
Discovery
Arbor and Dussoix in 1962 discovered that
certain bacteria contain Endonucleases which
have the ability to cleave DNA.
In 1970 Smith and colleagues purified and
characterized the cleavage site of a Restriction
Enzyme.
Werner Arbor, Hamilton Smith and Daniel
Nathans shared the 1978 Nobel prize for
Medicine and Physiology for their discovery of
Restriction Enzymes.
Biological Role
Most bacteria use Restriction Enzymes as a
defence against bacteriophages.
Restriction enzymes prevent the replication of
the phage by cleaving its DNA at specific sites.
The host DNA is protected by Methylases which
add methyl groups to adenine or cytosine bases
within the recognition site thereby modifying the
site and protecting the DNA.
Location of
methylase
Examples
site
Type I
Random
Around 1000bp
away from
recognition site
Endonuclease
and methylase
located on a
single protein
molecule
EcoK I
EcoA I
CfrA I
Type II
Specific
Within the
recognition site
Endonuclease
and methylase
are separate
entities
EcoR I
BamH I
Hind III
Type III
Random
24-26 bp away
Endonuclease
and methylase
EcoP I
Hinf III
5 G A T A T C 3
3 C T A T A G 5
Blunt Ends
CCCGGG
GGGCCC
Xma I
CCCGGG
GGGCCC
Sma I
Mechanism of Action
Restriction Endonuclease scan the length of the
DNA , binds to the DNA molecule when it
recognizes a specific sequence and makes one
cut in each of the sugar phosphate backbones of
the double helix by hydrolyzing the
phoshphodiester bond. Specifically,the bond
between the 3 O atom and the P atom is
broken.
Restriction Enzymes
can be used to
generate a restriction
map. This can provide
useful information in
characterizing a DNA
molecule.
Uses.
Restriction Fragment Length Polymorphism is a tool to study
variations among individuals & among species
Uses.