Restriction Enzymes

• What are restriction enzymes?

 Restriction Enzyme
• Enzyme that cuts DNA at specific nucleotide
sequences known as restriction sites.
• Found in bacteria and have evolved to provide
a defense mechanism against invading viruses.
• In bacteria they selectively cut up foreign DNA
in a process called restriction
• To cut the DNA, restriction enzyme makes two
incisions, each strand of the DNA double helix.
 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.
 Types of Restriction Enzymes
Cleavage Location of Examples
site methylase
Type I Random Endonuclease EcoK I
Around 1000bp and methylase EcoA I
away from located on a CfrA I
recognition site single protein
molecule
Type II Specific Endonuclease EcoR I
Within the and methylase BamH I
recognition site are separate Hind III
entities

Type III Random Endonuclease EcoP I

24-26 bp away and methylase Hinf III
from recognition located on a EcoP15 I
site single protein
molecule
 Restriction Enzymes
• Over 3000 have been identified
• More than 600 available commercially
• Routinely used for DNA modification and
manipulation in laboratories.
• http://en.wikipedia.org/wiki/Restriction_enzyme
Recognition sites of most restriction enzymes
have a twofold rotational symmetry

Restriction enzymes have corresponding symmetry to facilitate

recognition and usually cleave the DNA on the axis of symmetry
 Isoschizomers and Neochischizomers

• Restriction enzymes that have the same recognition sequence as well as

the same cleavage site are Isoschizomers.

• Restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence are Neochizomers.
Eg:SmaI and XmaI

CCCGGG CCCGGG
GGGCCC GGGCCC
Xma I Sma I
• Restriction Enzymes scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut
Restriction enzymes recognize and
make a cut within specific
palindromic sequences, known as
restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.
Each of the double
strands of the DNA
molecule is
complimentary to the
other; thus adenine
pairs with thymine,
and guanine with
cytosine.
 Hae III
HaeIII is a restriction enzyme that
searches the DNA molecule until it finds
this sequence of four nitrogen bases.

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site was found
HaeIII could go to work cutting
(cleaving) the DNA

5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
These cuts produce what scientists call
“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’
 “blunt ends” and “sticky ends”
Remember how HaeIII produced a “blunt end”?
EcoRI, for instance, makes a staggered cut and
produces a “sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’
blunt end

sticky end
Some more examples of restriction sites of
restriction enzymes with their cut sites:

HindIII: 5’ AAGCTT 3’
3’ TTCGAA 5’

BamHI: 5’ GGATCC 3’
3’ CCTAGG 5’

AluI: 5’ AGCT 3’
3’ TCGA 5’
 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.
Direct hydrolysis by nucleophilic attack at the
phosphorous atom

3’OH and 5’ PO43- is produced. Mg2+ is required for the catalytic activity of
the enzyme. It holds the water molecule in a position where it can attack
the phosphoryl group and also helps polarize the water molecule towards
deprotonation .
 The names for restriction enzymes come from:

• the type of bacteria in which the enzyme is found

• the order in which the restriction enzyme was identified
and isolated.

EcoRI for example

R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.
Structure of EcoR V endonuclease

• Consists of two subunits –

dimers related by two fold
rotational symmetry.
• Binds to the matching
symmetry of the DNA
molecule at the restriction
site and produces a kink at
the site.
Hydrogen bonding interactions between EcoRv and its DNA
substrate
A comparison of cognate and non-specific DNA in the
EcorV-DNA complex.
 Uses of Restriction Enzymes

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….

Restriction enzymes are

most widely used in
recombinant DNA
technology.
Restriction enzymes recognize and make a cut
within specific palindromic sequences,
known as restriction sites, in the genetic
code. This is usually a 4- or 6 base pair
sequence.

Example?
 Picking a palindrome
Words that read the same forwards as
backwards

Hannah hannaH

Level leveL

Madam madaM
 Palindromes in DNA sequences
Genetic palindromes
5 3’ are similar to verbal
’
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
3’ 5
’ sequence is identical
on both strands.
Separating Restriction Fragments, I
Separating Restriction Fragments, II
 References

• Biochemistry (1995), Wiley & Sons, Inc.

Voet D. and Voet J.G.
• Biochemistry (2002), Freeman & Co.
Berg, J.M., Tymoczco, J.L., Stryer, L.
• An Introduction to Genetic Analysis (2000), Freeman & Co.
Griffiths, A., Miller, J.H., Suzuki, D.T., Lewontin, R.C., Gelbart, W.M.
• Molecular Cell Biology (2000), Freeman & Co.
Lodish, Berk, Zipursky, Matsudaria, Baltimore, Darnell