1.

Introduction

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at or near
specific recognition nucleotide sequences known as restriction sites [1][2][3]. Restriction
enzymes are commonly classified into three types, which differ in their structure and whether
they cut their DNA substrate at their recognition site or if the recognition and cleavage sites
are separate from one another. To cut DNA, all restriction enzymes make two incisions, once
through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are
available commercially[4].These enzymes are routinely used for DNA modification in
laboratories, and they are a vital tool in molecular cloning[5][6][7].

2.History of restriction enzymes

In the early 1950s, a number of research teams observed differences in the efficiency of
bacteriophage infection on different bacterial host strains of the same species[8,9]. This was
described by Grasso and Paigen: when phage λ propagated in one strain of bacteria (e.g., E.
coil C) was used to infect another strain of the same species of bacteria (e.g., E. coil K), a
marked decrease in the rate of infection was noted compared to re-infection of the host strain
(E. col. i C). The new host (E. coil K) seemed to select against or "restrict" the incoming
phage. The researchers also noted this was not a hereditary phenomenon, because the phage
that did grow on the new strain could infect that strain at more typical rates after one round of
infection. The observed phenomenon was defined as "host control variation" and became an
area of intense research to discover the underlying mechanisms [10].

It was not until the 1960s that mechanisms underlying host control variation were determined
to involve enzymatic cleavage of the phage DNA, which led to the discovery and isolation of
restriction enzymes. In the early 1960s, Werner Artier observed that the host-range
determinant resided on the phage DNA, and subsequent experiments showed that methionine
[11]
was involved in host protection . These findings ultimately led to the proposal of a
restriction-modification (R-M) system, in which a restriction enzyme and a methylase from
the host work together to cleave foreign viral (non-methylated) DNA while keeping the
host DNA protected -through methylation[12]. Interestingly, most of the early work on R-M

1
systems was on Type I and III groups of restriction enzymes, classified based on
aspects of their structure and function (see Restriction enzyme classification).
However, the complete utility of restriction enzymes did not become apparent until
Kent Wilcox and Hamilton Smith discovered HindII,the first restriction enzyme of the
Type II class[13]. Hindi" recognizes a specific symmetrical DNA sequence and cleaves in
a defined manner within that recognition sequence. This feature, found in most early
Type II restriction enzymes, led Kathleen Danna and Daniel Nathans to use Hindi in
the physical mapping of simian virus 40 DNA [14], a process known as restriction
enzyme mapping.

For their pioneering work with restriction enzymes, Daniel Nathans, Hamilton Smith,
and Werner Arber were awarded the 1978 Nobel Prize in Physiology or Medicine.
With the discovery of DNA ligase, in combination with the growing family of site-
specific cutting restriction enzymes, recombinant DNA technology was born.
3.Origin

Restriction enzymes likely evolved from a common ancestor and became

widespread via horizontal gene transfer. In addition, there is mounting evidence that
restriction endonuclease evolved as a selfish genetic element.
4. Nomenclature

Since their discovery in the 1970s, many restriction enzymes have been
identified; for example, more tha n 3500 diffe re nt T ype II re stric tion
enzymes have been characterized [15]. Each enzyme is named after the bacterium from
which it was isolated, using a naming system based on bacterial genus, species and strain[16]
[17]
. For example, the name of the EcoRI restriction enzyme was derived as shown in the
box.

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Table - 1 Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia Genus

Co Coli Specific species

R RY13 Strain

I First Identified Order of identification in the bacterium

in the bacterium

Nomenclature of restriction endonucleases follows a general pattern :

(1) The first letter of the name of genus in which a given enzyme is
discovered is written in capital.

(2)This is followed by the first two letters of species name of the

organism. These three letters are generally written in italics, e.g., Eco
from Escherichia Coli, Hin from Haemophilusintluenzae, Hpa from
Haemophilusparainfluenzae, etc.
(3)Strain or type identification is depicted as subscript, e. g., Ecok, if the
enzyme is encoded by a plasmid, the plasmid name is written as a
subscript, e. g., EcoRI.
(4)When an organism produces more than one enzyme, they are
identified by sequential Roman numerals, e.g., the different enzymes
produced by H. influenza strain Rd are named Hindi], Hinc1.111, etc.

(5) All restriction enzymes in are designated by the general symbol R,

which is prefixed to their names, e.g., REcoRl_ RHindIII, RBamH1, etc. ( this
is to distinguish them from the corresponding methylases Isolated
from the same strains; the methylases are prefixed by M.
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5. Recognition site

A palindromic recognition site reads the same on the reverse strand as it does on the
forward strand when both are read in the same orientation. Restriction enzymes
recognize a specific sequence of nucleotides and produce a doublestranded cut in
the DNA. The recognition sequences usually vary between 4 and 8 nucleotides,
and many of them are palindromic, meaning the base sequence reads the same
backwards and forwards. In theory, there are two types of palindromic sequences that
can be possible in DNA. The mirror-like palindrome is similar to those found in
ordinary text, in which a sequence reads the same forward and backwards on a
single strand of DNA strand, as in GTAATG. The inverted repeat palindrome is also a
sequence that reads the same forward and backwards, but the forward and backward
sequences are found in complementary DNA strands (i.e., of double-stranded
DNA), as in GTATAC (GTATAC being complementary to CATATG). Inverted
repeat palindromes are more common and have greater biological importance than
minor-like palindromes.

EcoRI digestion produces "sticky" ends:

Whereas Srnal restriction e8nzyme cleavage produces "blunt"

ends:

Recognition sequences in DNA differ for each restriction enzyme,

producing differences in the length, sequence and strand orientation (5' end or the 3'
end) of a sticky-end "overhang"of an enzyme restriction. Different restriction enzymes
that recognize the same sequence are known as neoschizomers. These often cleave in
different locales of the sequence. Different enzymes that recognize and cleave in the
same location are known as isoschizomers.
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 6. Mechanism of Action of Restriction Enzymes:

Figure 1 : Steps in formation of recombinant DNA by the action of restriction Endonuclease

enzyme.

Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites,
but between the same two bases on the opposite strands. This leaves single-stranded portion at
the ends. There are overhanging stretches called sticky ends on each strand as given in above
figure.

Restriction endonucleases are also used in genetic engineering to form recombinant

molecules of DNA, which are composed of DNA from different sources or genomes. The
resultant DNA fragments have the same sticky ends, which are complementary to each other,
therefore can be joined together (end-to-end) using DNA ligases, when cut by the same
restriction enzyme.
In order to create a recombinant vector molecule, it is necessary that the vector and the source
DNA should cut with the same restriction enzyme.

7. Types of Restriction Endonuclease

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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[18][19][20] DNA sequence analyses of restriction enzymes however show
great variations, indicating that there are more than four types [21]. All types of enzymes
recognize specific short DNA sequences and carry out the endonucleolytic cleavage of
DNA to give specific fragments with terminal 5'-phosphates. They differ in their
recognition sequence, subunit composition, cleavage position, and cofactor
requirements[22][23] as summarized below:

 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
digestion and methylase activities.

 Type II enzymes cleave within or at short specific distances from a recognition site;
most require magnesium; single function (restriction digestion) enzymes
independent of methylase.

 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.
 Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and
glucosyl-hydroxymethylated DNA.
 Type V enzymes can target specific non-palindromic sequences and cut DNA of
variable length .
Type I

Type I restriction enzymes were the first to be identified and were first identified in two
different strains (K-12 and B) of E. coli. These enzymes cut at a site that differs, and is a
random distance (at least 1000 bp) away, from their recognition site by a process called DNA
Translocation. The recognition site is asymmetrical & composed of two specific portions, one
containing 3-4 nucleotides and another containing 4-5 nucleotides and are separated by a
non-specific spacer of about 6-8 nucleotides.
Types II
6
Type II enzyme cut DNA at defined positions close to or within their recognition sequences.
They produce discrete restriction fragments and distinct gel banding patterns, and they are the
only class used in the laboratory for routine DNA analysis and gene cloning. Rather
than forming a single family of related proteins, Type II enzymes are a collection of
unrelated proteins of many different sorts. Type II enzymes frequently differ so
completely in amino acid sequence from one another, and indeed from every other
known protein, that they exemplify the class of rapidly evolving proteins that are often
indicative of involvement in host-parasite interactions.

The most common Type II enzymes are those like Mai, HindIII and NotI that cleave DNA
within their recognition sequences. Enzymes of this kind are the principal ones available
commercially. They tend to be small, with subunits in the 200-350 amino acid range.

Type III

Type III enzymes are also large combination restriction-and-modification enzymes. They
cleave outside of their recognition sequences and require two such sequences in opposite
orientations within the same DNA molecule to accomplish cleavage; they rarely
give complete digests.

Type IV

Type IV enzymes recognize modified, typically methylated DNA and are exemplified by
the McrBC and Mrr systems of E. coli.

Type V

Type V restriction enzymes (e.g., the cas9-gRNA complex from CRISPRs) utilize

guide RNAs to target specific non-palindromic sequences found on invading
organisms. They can cut DNA of variable length, provided that a suitable guide RNA
is provided. The flexibility and ease of use of these enzymes make them promising for
future genetic engineering applications.

8. Applications of Restriction Endonuclease

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8.1 Recombinant DNA technology :

Recombinant DNA (rDNA) molecules are DNA molecules tbrmed by laboratory methods of
genetic recombination (such as molecular cloning) to bring together genetic material from
multiple sources, creating sequences that would not otherwise be found in the genome.

Restriction enzyme in recombinant DNA technology :

Figure 2: Restriction enzyme in recombinant DNA technology

1. The restriction enzyme find out the recognition site and cuts within the palindrome
sequence but in a pair of staggered cuts between the G and the A nucleotides.

2. This staggered cut leaves a pair of identical single-stranded "sticky ends." The ends are
called sticky because they can hydrogen bond (stick) to a complementary sequence.
Figure 2 shows EcoRI making a single cut in a circular DNA molecule such as a
plasmid: the cut opens up the circle, and the linear molecule formed has two sticky ends.
Production of these sticky ends is another feature of restriction enzymes that
makes them suitable for recombinant DNA technology.

8
3. When two such fragments of DNA cut by the same restriction enzyme come together ,
they can join by base pairing .

4. The joined fragments will usually form either a linear moleculer or a circular one, as shown
here for a plasmid other combination of fragments can also occur.

5 . The enzyme DNA ligase then work as glue and bind them .

Figure 3:Role of restriction enzyme in recombinant DNA technology

8.2 Construction of Restriction Map :

Restriction map is a diagram or map of DNA molecule of an organism that shows specific site of
cleavage (Restriction site)

 Construction of restriction map was one of the first described uses of restriction
enzyme.
9
 vRestriction maps are used to identify the fragment of DNA which contain specific
genes.

vThe data from many restriction digest of a common DNA sample is combined to produce a
complete and accurate restriction map.

Restriction enzyme in Restriction Map :

EcoRI recognizes the sequence G A A T T C in double stranded DNA. This recognition
sequence is a palindrome with a two-fold axis of symmetry, because reading from 5' to 3'
on either strand of the helix gives the same sequence. The palindromic nature of the
restriction site is more obvious in the figure below. The dot in the center of the
restriction site denotes the axis of symmetry. EcoRI catalyzes the hydrolysis of the
phosphodiester bonds between G and A on both DNA strands. The restriction fragments
generated in the reaction have short single-stranded tails at the 5'-ends. These ends are
often referred to as "sticky ends," because of their ability to form hydrogen bonds with
complementary DNA sequences.

Figure 4:EcoRI catalyzes the cleavage of a palindrome recognition site.

10
 Figure 5: An illustration of construction of restriction map

8.3 Construction of DNA Fingerprint:

DNA Fingerprinting is the technology which is used to identify individuals on the basis ()lithe
molecular characteristics of the DNA.

vDNA fingerprint is a forensic technique used to identify individuals based on the

variations in their DNA sequences.

vDNA fingerprinting with Restriction endonuclease is actually an extended version of

DNA restriction maps.

v Variation in the DNA fingerprint can be can be used to identify individuals in a large
heterogeneous population.

vBy the combination of PCR techniques, even the tiny DNA sample obtained from the site
of crime can be used for generating fingerprint.

v It is extensively used in solving paternity disputes, identification of suspects in

forensic science

Restriction enzyme in Restriction Map:

DNA fingerprinting involves the electrophoretic analysis of DNA fragment sizes generated
by restriction enzymes. Restriction enzymes are endonucleases which catalyze the cleavage
of phosphodiester bonds within both DNA strands (Figure 7). The sites of cleavage occur in
or near very specific palindromic sequences of bases called recognition sites, which are
generally 4 to 8 base pairs in length. The two most commonly used restriction enzymes for
DNA profile analysis are Hae III and Hinf I, which are 4-base and 5-base cutting enzymes.
The examples in the figure 2 show recognition sites for various restriction enzymes. DNA
fingerprinting involves the electrophoretic analysis of DNA fragment sizes generated by
restriction enzymes. Restriction enzymes are endonucleases which catalyze the cleavage of
phosphodiester bonds within both DNA strands. The sites of cleavage occur in or near very

11
specific palindromic sequences of bases called recognition sites, which are generally 4 to 8
base pairs in length. The two most commonly used restriction enzymes for DNA profile
analysis are Hae III and Hinf I, which are 4-base and 5-base cutting enzymes (Figure 6). The
examples in the figure 6 show recognition sites for various restriction enzymes.

Figure 6 : Restriction enzyme recognition sites

12
Figure 7 : Restriction fragments are created by Restriction enzyme.

13
 Figure 8 : An illustration of DNA fingerprinting procedure.

8.4 DNA cloning :

DNA cloning is the process of making multiple, identical copies of a particular piece
of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of
interest is first inserted into a circular piece of DNA called a plasmid. The
insertion is done using enzymes that "cut and paste" DNA, and itproduces a
molecule of recombinant DNA, or DNA assembled out of fragments from multiple
sources.

A circular piece of plasmid DNA has overhangs on its ends that match those of a
gene fragment. The plasmid and gene fragment are joined together to produce a
gene-containing plasmid. This gene-containing plasmid is an example of
recombinant DNA, or a DNA molecule assembled from DNA from multiple
sources.

Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the
plasmid are selected and grown up. As they reproduce, they replicate the plasmid and
pass it on to their offspring, making copies of the DNA it contains.

14
 Figure 9: DNA cloning overview .

Mechanism of restriction enzyme in DNA cloning :

When EcoRI recognizes and cuts this site, it always does so in a very specific
pattern that produces ends with single-stranded DNA "overhangs". If another piece of
DNA has matching overhangs (for instance, because it has also been cut by EcoRI),
the overhangs can stick together by complementary base pairing. For this reason,
enzymes that leave single-stranded overhangs are said to produce sticky ends. Sticky
ends are helpful in cloning because they hold two pieces of DNA together so they
can be linked by DNA ligase.

Figure 10: EcoRI cuts the DNA into many

fragments.
9. Conclusion

In conclusion, restriction enzymes are used to cut DNA into fragments, which is necessary
for DNA recombination and electrophoresis. DNA recombination in this lab used
restriction enzymes to insert ampicillin resistant genes into bacterial cells using bacterial
plasmids, which take up the genes. Heat shock was used to make the bacterial plasma
membrane permeable to the recombinant DNA, allowing it to pass through the
membrane. DNA electrophoresis is used to determine the size of the fragments that are

15
cut by restriction enzymes. Restriction enzymes only cut at their specific protein
recognition sites.

In this era , restriction enzyme is like a miracle . With the help of it we have gone one
step forward . In the following time hope it will make our world easier more than ever.

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