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Restriction Enzymes
(Restriction Endonucleases)
(NOTES)

Restriction enzyme is a protein (nuclease) that recognizes a specific, short

nucleotide sequence of DNA (known as restriction site or target sequence or
recognition sequence) and then cuts the DNA only at that specific site.

Restriction enzymes are found in bacteria (and other prokaryotes). Each

restriction enzyme recognizes just one or a few restriction sites. After recognizing
its target sequence, a restriction enzyme makes a double-stranded cut in the DNA
molecule. Typically, the cut is at or near the restriction site and occurs in an
orderly, predictable pattern.

RESTRICTION SITE
(4-6 bp long)

RESTRICTION ENZYME
(Molecular scissors)

More than 400 restriction enzymes have been isolated from the bacteria that
manufacture them. In live bacteria, restriction enzymes function to defend the
cell against invading viral bacteriophages. Restriction enzymes of the bacteria
cleave the restriction sites in the viral genome, fragmenting and destroying the
DNA of invading bacteriophages before it can incorporate into the host's genome
(bacterial genome) and take over the cell.
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Interestingly, a bacterium is immune (resistant) to its own restriction enzymes,

even if it has the target sequences, which could be destroyed by its own
restriction enzymes. This is because the bacterial restriction sites are highly
methylated, making them unrecognizable to the restriction enzymes.

Methylase enzyme adds a methyl groups

(—CH3) to adenine or cytosine bases
within the recognition sequence of DNA,
which is, thus, modified and protected
from the own endonucleases of the
bacterium. The restriction enzyme and its
corresponding methylase constitute the
restriction-modification system of a ↑
bacterial species. Methylation protection of
restriction sites of bacterial genome

Methylation protection of restriction

sites of bacterial genome →

Methyl group (-CH3) is transferred from S-adenosyl methionine (SAM) in a reaction

catalyzed by DNA methyl transferase (DNMT) or methylase. SAM is converted to SAH
(S-adenosyl homocysteine)
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Nature of cleavage by restriction endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable
importance.
They cut the DNA molecule in two ways:
(1) Many restriction endonucleases cleave both strands of DNA simply at the
same point within the recognition sequence. As a result of this type of cleavage,
the DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the
examples of restriction endonucleases producing blunt ends. Blunt ends may also
be referred to as protruding ends.

(2) In the other style of cleavage by the restriction endonucleases, the two
strands of DNA are cut at two different points. Such cuts are termed as staggered
cuts and this results into the generation of protruding ends i.e., one strand of the
double helix extends a few bases beyond the other strand. Such ends are, called
cohesive or sticky ends.
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Types of DNA cuts generated by restriction enzyme EcoR1

The following Table shows sticky and blunt ends generated in the restriction sites
by various restriction enzymes.

Enzyme Source Recognition Sequence Cut type (Sticky or Blunt)

Escherichia coli 5'GAATTC 5'---G AATTC---3'

EcoR1
Sticky ends 3'CTTAAG 3'---CTTAA G---5'

Bacillus amyloliquefaciens 5'GGATCC 5'---G GATCC---3'

BamH1
Sticky ends 3'CCTAGG 3'---CCTAG G---5'

Thermus aquaticus 5'TCGA 5'---T CGA---3'

Taq1
Sticky ends 3'AGCT 3'---AGC T---5'

Arthrobacter luteus 5'AGCT 5'---AG CT---3'

Alu1
Blunt ends 3'TCGA 3'---TC GA---5'
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Examples: As an example of how a restriction enzyme recognizes and cuts at a

DNA sequence, let us consider EcoRI, a common restriction enzyme used in
biotechnology labs. The EcoRI cuts at the following restriction site:

If we place the two DNA strands together in a straight line, they appear as
palindromes (mirror images)

5'-...GAATTC...-3' 3'-...CTTAAG...-5'

A palindromic sequence is a nucleic acid sequence on double-

stranded DNA or RNA, wherein reading 5'-end to 3'-end forward on one strand
matches the sequence reading 3'-end to 5'-end on the complementary strand with
which it forms a double helix.

The top strand reads 5'-GAATTC-3', while the bottom strand reads 3'-CTTAAG-5'. If
the DNA strand is flipped over, the sequences are exactly the same ( 5'GAATTC-3' and
3'-CTTAAG-5').

An EcoRI enzyme binds to an EcoRI site in a piece of DNA and makes a cut on both
strands of the DNA as shown below using arrows.

5'-...G↓AATTC...-3' 3'-...CTTAA↓G...-5'

When EcoRI recognizes and cuts its restriction site, it always does so in a very
specific pattern that produces ends with single-stranded DNA overhangs. Thus, it
produces an overhang of 5'-AATT-3' on each end of the cut DNA. The pattern of
the cut is called as staggered cut, which produce sticky ends as shown below:
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A staggered cut in DNA

DNA - 1 DNA - 2
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The two sticky ends may join together by complementary base pairing. If another
piece of DNA (foreign DNA) has matching overhangs (it can occur when both
DNAs are cut with the same restriction enzymes such as EcoRI), the overhangs of
the two different DNA-stretches can also stick together by complementary base
pairing. For this reason, enzymes that leave single-stranded overhangs are said to
produce highly reactive sticky ends on the broken ends. Sticky ends are helpful in
cloning and genetic engineering because they hold two pieces of DNA together so
they can be linked by DNA ligase enzyme.

Not all restriction enzymes produce sticky ends. Some are blunt cutters, which cut
straight down the middle of a target sequence and leave no overhangs. The
restriction enzyme SmaI is an example of a blunt cutter. See below:

A blunt cut in DNA

A SmaI enzyme binds to the SmaI restriction site, which is given below:

5'-...CCCGGG...-3' 3'-...GGGCCC...5'
It makes a cut right in the middle of this sequence on both strands, producing
blunt ends. The cut sites are shown below:

5'-...CCC↓GGG...-3' 3'-...GGG↓CCC...5'
Blunt-ended DNA fragments can also be joined to each other by DNA ligase.
However, blunt-ended fragments are harder to ligate (join) together (the ligation
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reaction is less efficient and more likely to fail) because there are no single-
stranded overhangs to hold the DNA molecules in position.

DNA ligase is the molecular glue or gum, which joins together the cut-ends of DNA (sticky or
blunt) by creating phosphodiester bonds necessary to complete the sugar-phosphate backbone
of the newly synthesized transgenic DNA.

Phosphodiester bond in DNA Phosphodiester bond in DNA and RNA

Types of Restriction Endonucleases:

There are 3 main categories of restriction Endonuclease enzymes:

1. Type-I
2. Type-II
3. Type-III

Type-I Restriction Endonucleases:

These are the complex type of endonucleases which cleave only one strand of
DNA. These enzymes have the recognition sequences of about 15 bp length. They
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require Mg++ ions and ATP for their functioning. Such types of restriction
endonucleases cleave the DNA about 1000 bp away from the 5′-end of the
sequence TCA located within the recognition site. Important examples of Type-I
restriction endonuclease enzymes are EcoK, EcoB, etc.

Type-II Restriction Endonucleases:

These are most important endonucleases for gene cloning and hence for
Recombinant DNA Technology. These enzymes are most stable. They show
cleavage only at specific sites and therefore they produce the DNA fragments of a
defined length. These enzymes show cleavage in both the strands of DNA,
immediately outside the recognition sequences. They require Mg++ ions for their
functioning.

Such enzymes are advantageous because they do not require ATP for cleavage
and they cause cleavage in both strands of DNA. Only Type II Restriction
Endonucleases are used for gene cloning due to their suitability.

The recognition sequences for Type-II Restriction Endonuclease enzymes are in

the form of palindromic sequences with rotational symmetry, i.e., the base
sequence in the first half of one strand of DNA is the mirror image of the second
half of other strand of that DNA double helix. Important examples of Type-II
Restriction endonucleases include Hinfl, EcoRI, PvuII, Alul, Haelll, etc.

palindromic sequences with rotational symmetry

Type-III Restriction Endonucleases

These restriction enzymes are not used for gene cloning. They are the
intermediate enzymes between Type-I and Type-II restriction endonuclease. They
require Mg++ ions and ATP for cleavage and they cleave the DNA at well-defined
sites in the immediate vicinity of recognition sequences, e.g. Hinf III, etc.
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Nomenclature of restriction enzymes

Restriction enzymes are named based on the organism in which they were
discovered. For example, the enzyme Hind III was isolated from the bacterium
Haemophilus influenzae, strain Rd. The first three letters (Hin) of the name are
italicized because they abbreviate the genus (H) and species (in) names of the
organism. The fourth letter (d) typically comes from the bacterial strain
designation. Typically, the Roman numerals (I, II, III, etc.) indicates the order in
which restriction enzymes were discovered in a particular strain.

Nomenclature of restriction enzymes consists three parts:

 It indicates the genus and species of specific organism by using

abbreviation into three letters.

 It indicates the strain of the relevant species by using a letter, number or its
combination.

 It indicates specific restriction modification systems present in the same

organism or strain by using a roman numeral.
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Example: the name of EcoRI restriction enzyme is derived as:

Abbreviation Corresponding full form

E Escherichia (genus)
Co coli (species)
R RY13 (strain)
I First identified in the bacterium

Exonucleases:
Exonuclease is an enzyme that removes nucleotides from the ends of a nucleic
acid molecule. An exonuclease removes nucleotide from the 5′ or 3′ end of a DNA
molecule. An exonuclease never produces internal cuts in DNA.
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EXONUCLEASE ACTIVITY →

In recombinant DNA technology, various types of exonucleases are employed like

Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc.