Restriction enzymes genetic enginering

Also referred to as Restriction Endonucleases

Molecular scissors that cut double stranded DNA molecules at specific points.

Found naturally in a wide variety of prokaryotes

An important tool for manipulating DNA.

Enters and recognizes a certain sequence on a double helix strand of DNA, usually 4-6 base-pairs long, and cuts it.

Precise by cutting both strands in same location though strands move in reverse directions; REs are able to depict the precise spot to cut

2. RESTRICTION ENDONUCLEASES

3. What are restriction enzymes?
•Also referred to as Restriction Endonucleases
•Molecular scissors that cut double stranded DNA molecules at
specific points.
•Found naturally in a wide variety of prokaryotes
•An important tool for manipulating DNA.
•Enters and recognizes a certain sequence on a double helix strand
of DNA, usually 4-6 base-pairs long, and cuts it.

•Precise by cutting both strands in same location though strands
move in reverse directions; REs are able to depict the precise spot
to cut

4. •Able to restrict and destroy foreign DNA, such as viruses,
preventing them from entering the cell
•Used in biotechnology for cutting DNA into smaller strands for
research in gene cloning or fragment lengths among different
individuals

5. •Discovery
•The term restriction enzyme originated from the studies of phage λ.


•In 1960 it was shown by werner arber and matthew meselson but
theystudied only about type 1 restriction enzymes.
•In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated and
characterized the first type II.
•restriction enzyme, HindII, from the bacterium Haemophilus influenza.For
their work in the discovery and characterization of restriction enzymes, the
1978 Nobel Prize for Physiology or Medicine was awarded to Werner Arber,
Daniel Nathans, and Hamilton O. Smith

6. Dr. Nathans Dr. Daniel Nathans with colleague, Dr. Hamilton Smith

7. Daniel Nathans and Kathleen Danna Experiment
Digested DNA from plague purified stocks SV40 with the restriction
endonuclease Hemophillus influenza.
Provided eleven fragments that were resolvable by polyacrylamide gel
electrophoresis.
Eight of which were equimolar to the original DNA.
Fragments ranged from 6.5 × 105 to 7.4 × 104 daltons which was determined
by electron microscopy, DNA content, or electrophoretic mobility

8. 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.

10. Inside a prokaryotic , the restriction enzymes selectively cut up
foreign DNA in a process called restriction .
while host DNA is protected by modification enzyme ( a
methyltransferase ) that modifies the prokaryotic DNA and blocks
cleavage.
Together , these two processes from the restriction modification
system .

13. Types of Restriction Enzymes
Cleavage

site




Examples
Type I Random
Around 1000bp
away from
recognition site
EcoK I
EcoA I
CfrA I
Type II Specific
Within the
recognition site
EcoR I
BamH I
Hind III
Type III Random
24-26 bp away
from recognition
site
EcoP I
Hinf III
EcoP15 I

14. Types of restriction enzymes

Naturally occurring restriction endonucleases are categorized into four groups
(Types I, II III, and IV) based on
their composition and enzyme cofactor requirements, the nature of their
target sequence, and the position of their DNA cleavage site relative to the
target sequence
◘ Type I enzymes
cleave at sites remote from a recognition site ○require both ATP and S-
adenosyl-L-methionine to function ○ multifunctional protein with both restriction
and methylase activities.
one enzyme with different subunits for recognition ,cleavage ,and methylation
,Recognizes and methylates a single sequence but cleaves DNA up to 1000 bp
away .

15. ◘ Type II enzymes
cleave within or at short specific distances from a recognition site; most
require magnesium; single function (restriction) enzymes independent of
methylase.
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 enzymes
cleave at sites a short distance from a recognition site; require ATP (but do
not hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not
required; exist as part of a complex with a modification methylase .
one enzyme with two different subunits , one for recognition and modification
and one for cleavage , recognition and methylates same sequence but cleaves
24-26 bp away .

16. ◘ Type IV enzymes
Enzymes target modified DNA, e.g. methylated, hydroxymethylated and
glucosyl-hydroxymethylated DNA.
Two different enzymes but recognition sequence is symmetric , cleavage
occurs in one side of recognition sequence up to 20 bp away .
◘ Nomenclature
Each enzyme is named after the bacterium from which it was isolated, using
a naming system based on bacterial genus, species and strain.
For example, the name of the EcoRI restriction enzyme was derived as
shown in the box.

18. ◘ Mechanism of Action of restriction Enzymes
The process is one of recognition of the binding site , binding of the enzyme
dimer to the DNA , cleavage of the DNA , and enzyme release .
To begin , all restriction endonucleases will bind DNA specifically and , with
much less strength , non-specifically .
It is probable that even non-specific DNA binding will induce a conformational
change in the restriction enzyme dimer that will result in the protein adapting to the
surface of the DNA strands .
The Homodimer will either bind directly to the recognition site(specific binding) or
nearby ( non- specific binding )
In the case of non-specific binding , if the recognition site is not too far away the
enzyme will move along the DNA strand until it hits the recognition site .
Once the enzyme locates the recognition site it will couple and then hydrolyze
the sugar phosphate bonds of the DNA .

19. Finally , the enzyme will release leaving the cleaved DNA molecule behind .
In general , intimate contact is held by 15 – 20 hydrogen bonds that form
between the protein and the DNA bases in the recognition site .
These bonds are shown to be mediated through specific amino acids ,
primarily aspartic acid or aspartate (Asp) and glutamic acid or glutamate
(Glu), held in a proper three-dimensional configuration

This requires significant conformational changes in both the protein and the
DNA as well as expulsion(removel) of water molecules from the p.

20. 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

21. Restriction fragments can be blunt ended or
sticky ended
5’ G A A T T C 3’ 5’ G A T A T C 3’
3’ C T T A A G 5’ 3’ C T A T A G 5’

Sticky Ends Blunt Ends
Sticky ends or blunt ends can be used to join DNA
fragments.
Sticky ends are more compared to blunt ends.

22. •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
C C C G G G C C C G G G
G G G C C C G G G C C C
Xma I Sma I
Isoschizomers and Neochischizomers

23. 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.

26. Uses of Restriction Enzymes
•Restriction Enzymes can
be used to generate a
restriction map.
•This can provide useful
information in
characterizing a DNA
molecule.

27. Uses….
Restriction Fragment Length Polymorphism is a tool to study
variations among individuals & among species

28. Uses….
Restriction enzymes are
most widely used in
recombinant DNA
technology.

29. •Laboratory Applications for Restriction Enzymes
•Provides different ways of manipulating DNA such as the creation of
recombinant DNA, which has endless applications
•Allows for the large scale production human insulin for diabetics using
E. coli, as well as for the Hepatitis B and HPV vaccines
•Cloning DNA Molecules
•Studying nucleotide sequence

30. Full Restriction Digest
• DNA at each restriction site creates multiple
restriction fragments:
Is it possible to reconstruct the order of the fragments from the
sizes of the fragments {3,5,5,9} ?

31. Measuring Length of Restriction Fragments
•Restriction enzymes break DNA into restriction fragments.
•Gel electrophoresis is a process for separating DNA by size
and measuring sizes of restriction fragments
•Can separate DNA fragments that differ in length in only 1
nucleotide for fragments up to 500 nucleotides long

32. Gel Electrophoresis
•DNA fragments are injected into a gel positioned in an
electric field
•DNA are negatively charged near neutral pH
•The ribose phosphate backbone of each nucleotide is acidic;
DNA has an overall negative charge
•DNA molecules move towards the positive electrode
•DNA fragments of different lengths are separated
according to size
–Smaller molecules move through the gel matrix more readily
than larger molecules
•The gel matrix restricts random diffusion so molecules
of different lengths separate into different bands

33. Gel Electrophoresis: Example
Direction of DNA
movement
Smaller fragments
travel farther
Molecular Cell Biology, 4
th
edition

34. Vizualization of DNA:
Autoradiography and Fluorescence
•autoradiography:
•The DNA is radioactively labeled. The gel is laid against a
sheet of photographic film in the dark, exposing the film at
the positions where the DNA is present
•fluorescence:
•The gel is incubated with a solution containing the
fluorescent dye ethidium – ethidium binds to the DNA
•The DNA lights up when the gel is exposed to ultraviolet
light.