Restriction Endonucleases
Definition
An enzyme from bacteria that can recognize specific base sequences in DNA and cut (restrict) the
DNA at that site (the restriction site) called a restriction enzyme.
OR
Restriction enzyme, also called restriction endonuclease, a protein produced
by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction
enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated
from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that
contain genes; for this reason they are indispensible tools of recombinant DNA technology (genetic
engineering).
Introduction
Restriction enzymes are also called ‘molecular scissors’ as they cleave DNA at or near specific
recognition sequences known as restriction sites. These enzymes make one incision on each of the two
strands of DNA and are also called restriction endonuclease.
Viruses infect the host cells by injecting their DNA into the cells. This viral DNA hijacks the host cell’s
machinery for reproduction of viral progeny, resulting in the host cell’s death. To overcome the viral
infection, many bacteria and Achaea have evolved several mechanisms. A major protective mechanism
involves the use of restriction enzymes to degrade the invading viral DNA by cleaving it at specific
restriction sites. At the same time, the host cell protects its own DNA from being cleaved by employing
other enzymes called methylase, which methylase adenine or cytosine bases within host recognition
sequences. For each of the restriction enzyme, the host cell produces a corresponding methylase that
methylates and protects the host DNA from degradation. These enzymes make up the restriction-
modification (R-M) systems.
The restriction enzymes catalyze the hydrolysis of the bond between the 3’-oxygen atom and the
phosphorus atom in the phosphodiester backbone of DNA. The enzymes require Mg2+ or other divalent
ions for their activity.
Discovery of Restriction Endonucleases
The term restriction enzyme originated from the studies of phage
In 1960 it was shown by Werner Arber and Matthew Meselson but they studied only about type I
restriction enzyme.
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.
Categorization of Restriction Enzymes on the basis of:-
Their composition.
Enzyme cofactor requirement.
The nature of their target sequence.
Position of their DNA cleavage site relative to the target sequence.
How Restriction Endonucleases work
Restriction enzymes recognize a specific sequence of nucleotides, and produce a double stranded cut in
the DNA. These cuts are of two types:
1. BLUNT END 2. STICKY END
CCC GGG G AATTC
GGG CCC CTTAA G
BLUNT END
These blunt ended fragments can be joined to any other DNA fragment with blunt ends.
Enzymes useful for certain types of DNA cloning experiments.
STICKY END
DNA fragments with complimentary sticky ends can be combined to create new molecules which
allow the creation and manipulation of DNA sequences from different sources.
Types of Restriction Enzymes
Restriction enzymes are categorized into three general groups.
Type I Restriction Endonucleases
Type II Restriction Endonucleases
Type III Restriction Endonucleases
Type I Restriction Endonucleases
Type I restriction enzymes are complex endonucleases, and have recognition
sequence of 15 bp; they cleave the DNA about 1000 bp away from the 5` end of
the sequence “TCA” located within the recognition site. Eg:- EcoK-12,EcoB,etc.
The type I Restriction Enzyme work with the help of many co factor like as SAdenosyl
Methionine, Hydrolysed Adenosine Triphosphate (ATP) ad Magnesium (Mg2+).
The type I Restriction Enzyme methylates the DNA sequence at the site of
reognition.The type I Restriction Enzyme translocate the DNA sequence.
Subunits of Type I Restriction Endonucleases
Type I restriction enzymes possess three subunits:
HsdR: is required for restriction.
HsdM: necessary for adding methyl groups to host DNA (methyltransferase activity).
HsdS: important for specificity of cut site recognition in addition to its methyltransferase activity.
Type II Restriction Endonucleases
The type II restriction enzymes are remarcably stable and induce cleavage either, in most cases,
within or outside their recognition sequences,which are symmetrical.
The type I Restriction Enzyme work with the help of only single co factor like as Magnesium
(Mg2+).
The Ist type II Enzyme to be isolated was Hind II in 1970.
Only type II restriction endonucleases are used for restriction mapping and gene cloning in view
of their cleavage only at specific site.
Cut of Type II Restriction Endonucleases
Type II restriction enzymes can generate two different types of cuts depending on whether they
cut both strands at the centre of the recognition sequence:
The former cut will generate “blunt ends” with no nucleotide over hangs.
The latter, generates “sticky” or “cohesive” ends
Subunits of Type II Restriction Endonucleases
These subgroups are defined using a letter suffix.
Type II B restriction enzymes.
Type II E restriction endonucleases.
Type II M restriction endonucleases.
Type II T restriction enzymes
Type III Restriction Endonucleases
Type III restriction enzymes are intermediate between type I and type II endonucleases, they
cleave DNA in the immediate vacinity of their recognition sites, Eg;- EcoP1, EcoP15, Hind III etc.
They cut DNA upto 20-30 base pairs away from the recognition site.
These enzymes contain more than one subunit.(ATP & Mg2+)
And require AdoMet and ATP cofactors for their roles in DNA methylation and
restriction.
Nomenclature
Recognition sequence of Restriction Endonucleases :-
The recognition sequence of Type II endonucleases form palindrome with rotational symmetry.
Mostly Type II endonucleases have recognition sites of 4,5 or 6 bp. Which are predominantly GC
rich.
Properties of Restriction Enzymes :-
Applications of Restriction Enzymes :-
They are used in gene cloning and protein expression experiments.
Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study
fragment length differences among individuals (Restriction Fragment Length Polymorphism –
RFLP).
Each of these methods depends on the use of agarose gel electrophoresis for separation of the
DNA fragments.
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.
