Restriction enzyme

A restriction enzyme, restriction

endonuclease, or restrictase is an enzyme
that cleaves DNA into fragments at or near
specific recognition sites within molecules
known as restriction sites.[1][2][3]
Restriction enzymes are one class of the
broader endonuclease group of enzymes.
Restriction enzymes are commonly
classified into five 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...

These enzymes are found in bacteria and

archaea and provide a defence
mechanism against invading viruses.[4][5]
Inside a prokaryote, the restriction
enzymes selectively cut up foreign DNA in
a process called restriction digestion;
meanwhile, host DNA is protected by a
modification enzyme (a
methyltransferase) that modifies the
prokaryotic DNA and blocks cleavage.
Together, these two processes form the
restriction modification system.[6]

Over 3,000 restriction enzymes have been

studied in detail, and more than 600 of
these are available commercially.[7] These
enzymes are routinely used for DNA
modification in laboratories, and they are a
vital tool in molecular cloning.[8][9][10]

History
The term restriction enzyme originated
from the studies of phage λ, a virus that
infects bacteria, and the phenomenon of
host-controlled restriction and
modification of such bacterial phage or
bacteriophage.[11] The phenomenon was
first identified in work done in the
laboratories of Salvador Luria, Weigle and
Giuseppe Bertani in the early 1950s.[12][13]
It was found that, for a bacteriophage λ
that can grow well in one strain of
Escherichia coli, for example E. coli C,
when grown in another strain, for example
E. coli K, its yields can drop significantly, by
as much as 3-5 orders of magnitude. The
host cell, in this example E. coli K, is
known as the restricting host and appears
to have the ability to reduce the biological
activity of the phage λ. If a phage
becomes established in one strain, the
ability of that phage to grow also becomes
restricted in other strains. In the 1960s, it
was shown in work done in the
laboratories of Werner Arber and Matthew
Meselson that the restriction is caused by
an enzymatic cleavage of the phage DNA,
and the enzyme involved was therefore
termed a restriction enzyme.[4][14][15][16]

The restriction enzymes studied by Arber

and Meselson were type I restriction
enzymes, which cleave DNA randomly
away from the recognition site.[17] In 1970,
Hamilton O. Smith, Thomas Kelly and Kent
Wilcox isolated and characterized the first
type II restriction enzyme, HindII, from the
bacterium Haemophilus influenzae.[18][19]
Restriction enzymes of this type are more
useful for laboratory work as they cleave
DNA at the site of their recognition
sequence and are the most commonly
used as a molecular biology tool.[20] Later,
Daniel Nathans and Kathleen Danna
showed that cleavage of simian virus 40
(SV40) DNA by restriction enzymes yields
specific fragments that can be separated
using polyacrylamide gel electrophoresis,
thus showing that restriction enzymes can
also be used for mapping DNA.[21] 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.[22] The discovery
of restriction enzymes allows DNA to be
manipulated, leading to the development
of recombinant DNA technology that has
many applications, for example, allowing
the large scale production of proteins such
as human insulin used by diabetics.[12][23]

Origins
Restriction enzymes likely evolved from a
common ancestor and became
widespread via horizontal gene
transfer.[24][25] In addition, there is
mounting evidence that restriction
endonucleases evolved as a selfish
genetic element.[26]

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[2] and produce a
double-stranded cut in the DNA. The
recognition sequences can also be
classified by the number of bases in its
recognition site, usually between 4 and 8
bases, and the number of bases in the
sequence will determine how often the site
will appear by chance in any given
genome, e.g., a 4-base pair sequence
would theoretically occur once every 4^4
or 256bp, 6 bases, 4^6 or 4,096bp, and 8
bases would be 4^8 or 65,536bp.[27] Many
of them are palindromic, meaning the base
sequence reads the same backwards and
forwards.[28] 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 backward on a
single strand of DNA, as in GTAATG. The
inverted repeat palindrome is also a
sequence that reads the same forward
and backward, 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).[29] Inverted repeat palindromes
are more common and have greater
biological importance than mirror-like
palindromes.

EcoRI digestion produces "sticky" ends,

whereas SmaI restriction enzyme 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 3' end) of a
sticky-end "overhang" of an enzyme
restriction.[30]

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.

Types
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.[31][32][33] DNA sequence
analyses of restriction enzymes however
show great variations, indicating that there
are more than four types.[34] 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,[35][36] as
summarised below:

Type I enzymes (EC 3.1.21.3 ) 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 (EC 2.1.1.72 ) activities.
Type II enzymes (EC 3.1.21.4 ) 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 (EC 3.1.21.5 ) 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 (EC 2.1.1.72 ).
 Type IV enzymes target modified DNA,
e.g. methylated, hydroxymethylated and
glucosyl-hydroxymethylated DNA

Type l …

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.[37] These enzymes cut at a site that
differs, and is a random distance (at least
1000 bp) away, from their recognition site.
Cleavage at these random sites follows a
process of DNA translocation, which
shows that these enzymes are also
molecular motors. The recognition site is
asymmetrical and is composed of two
specific portions—one containing 3–4
nucleotides, and another containing 4–5
nucleotides—separated by a non-specific
spacer of about 6–8 nucleotides. These
enzymes are multifunctional and are
capable of both restriction digestion and
modification activities, depending upon
the methylation status of the target DNA.
The cofactors S-Adenosyl methionine
(AdoMet), hydrolyzed adenosine
triphosphate (ATP), and magnesium
(Mg2+) ions, are required for their full
activity. Type I restriction enzymes
possess three subunits called HsdR,
HsdM, and HsdS; HsdR is required for
restriction digestion; HsdM is necessary
for adding methyl groups to host DNA
(methyltransferase activity), and HsdS is
important for specificity of the recognition
(DNA-binding) site in addition to both
restriction digestion (DNA cleavage) and
modification (DNA methyltransferase)
activity.[31][37]

Type II …
 Type II site-specific
deoxyribonuclease-like

Structure of the homodimeric restriction

enzyme EcoRI (cyan and green cartoon
diagram) bound to double stranded DNA
(brown tubes).[38] Two catalytic magnesium
ions (one from each monomer) are shown as
magenta spheres and are adjacent to the
cleaved sites in the DNA made by the enzyme
(depicted as gaps in the DNA backbone).

Identifiers

Symbol Restrct_endonuc-II-
 like
Pfam clan CL0236

InterPro IPR011335

SCOPe 1wte / SUPFAM

Typical type II restriction enzymes differ

from type I restriction enzymes in several
ways. They form homodimers, with
recognition sites that are usually undivided
and palindromic and 4–8 nucleotides in
length. They recognize and cleave DNA at
the same site, and they do not use ATP or
AdoMet for their activity—they usually
require only Mg2+ as a cofactor.[28] These
enzymes cleave the phosphodiester bond
of double helix DNA. It can either cleave at
the center of both strands to yield a blunt
end, or at a staggered position leaving
overhangs called sticky ends.[39] These are
the most commonly available and used
restriction enzymes. In the 1990s and
early 2000s, new enzymes from this family
were discovered that did not follow all the
classical criteria of this enzyme class, and
new subfamily nomenclature was
developed to divide this large family into
subcategories based on deviations from
typical characteristics of type II
enzymes.[28] These subgroups are defined
using a letter suffix.
Type IIB restriction enzymes (e.g., BcgI
and BplI) are multimers, containing more
than one subunit.[28] They cleave DNA on
both sides of their recognition to cut out
the recognition site. They require both
AdoMet and Mg2+ cofactors. Type IIE
restriction endonucleases (e.g., NaeI)
cleave DNA following interaction with two
copies of their recognition sequence.[28]
One recognition site acts as the target for
cleavage, while the other acts as an
allosteric effector that speeds up or
improves the efficiency of enzyme
cleavage. Similar to type IIE enzymes, type
IIF restriction endonucleases (e.g.
NgoMIV) interact with two copies of their
recognition sequence but cleave both
sequences at the same time.[28] Type IIG
restriction endonucleases (e.g., Eco57I) do
have a single subunit, like classical Type II
restriction enzymes, but require the
cofactor AdoMet to be active.[28] Type IIM
restriction endonucleases, such as DpnI,
are able to recognize and cut methylated
DNA.[28][40][41] Type IIS restriction
endonucleases (e.g., FokI) cleave DNA at a
defined distance from their non-
palindromic asymmetric recognition
sites;[28] this characteristic is widely used
to perform in-vitro cloning techniques
such as Golden Gate cloning. These
enzymes may function as dimers.
Similarly, Type IIT restriction enzymes
(e.g., Bpu10I and BslI) are composed of
two different subunits. Some recognize
palindromic sequences while others have
asymmetric recognition sites.[28]

Type III …

Type III restriction enzymes (e.g., EcoP15)

recognize two separate non-palindromic
sequences that are inversely oriented.
They cut DNA about 20–30 base pairs
after the recognition site.[42] These
enzymes contain more than one subunit
and require AdoMet and ATP cofactors for
their roles in DNA methylation and
restriction digestion, respectively.[43] They
are components of prokaryotic DNA
restriction-modification mechanisms that
protect the organism against invading
foreign DNA. Type III enzymes are hetero-
oligomeric, multifunctional proteins
composed of two subunits, Res (P08764)
and Mod (P08763). The Mod subunit
recognises the DNA sequence specific for
the system and is a modification
methyltransferase; as such, it is
functionally equivalent to the M and S
subunits of type I restriction
endonuclease. Res is required for
restriction digestion, although it has no
enzymatic activity on its own. Type III
enzymes recognise short 5–6 bp-long
asymmetric DNA sequences and cleave
25–27 bp downstream to leave short,
single-stranded 5' protrusions. They
require the presence of two inversely
oriented unmethylated recognition sites
for restriction digestion to occur. These
enzymes methylate only one strand of the
DNA, at the N-6 position of adenosyl
residues, so newly replicated DNA will
have only one strand methylated, which is
sufficient to protect against restriction
digestion. Type III enzymes belong to the
beta-subfamily of N6 adenine
methyltransferases, containing the nine
motifs that characterise this family,
including motif I, the AdoMet binding
pocket (FXGXG), and motif IV, the catalytic
region (S/D/N (PP) Y/F).[35][44]

Type IV …

Type IV enzymes recognize modified,

typically methylated DNA and are
exemplified by the McrBC and Mrr
systems of E. coli.[34]

Type V …

Type V restriction enzymes (e.g., the cas9-

gRNA complex from CRISPRs[45]) 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.[45][46]

Artificial restriction enzymes …

Artificial restriction enzymes can be

generated by fusing a natural or
engineered DNA binding domain to a
nuclease domain (often the cleavage
domain of the type IIS restriction enzyme
FokI).[47] Such artificial restriction enzymes
can target large DNA sites (up to 36 bp)
and can be engineered to bind to desired
DNA sequences.[48] Zinc finger nucleases
are the most commonly used artificial
restriction enzymes and are generally used
in genetic engineering
applications,[49][50][51][52] but can also be
used for more standard gene cloning
applications.[53] Other artificial restriction
enzymes are based on the DNA binding
domain of TAL effectors.[54][55]

In 2013, a new technology CRISPR-Cas9,

based on a prokaryotic viral defense
system, was engineered for editing the
genome, and it was quickly adopted in
laboratories.[56] For more detail, read
CRISPR (Clustered regularly interspaced
short palindromic repeats).

In 2017 a group from University of Illinois

reported using an Argonaute protein taken
from Pyrococcus furiosus (PfAgo) along
with guide DNA to edit DNA in vitro as
artificial restriction enzymes.[57]

Artificial ribonucleases that act as

restriction enzymes for RNA are also being
developed. A PNA-based system, called
PNAzymes, has a Cu(II)-2,9-
dimethylphenanthroline group that mimics
ribonucleases for specific RNA sequence
and cleaves at a non-base-paired region
(RNA bulge) of the targeted RNA formed
when the enzyme binds the RNA. This
enzyme shows selectivity by cleaving only
at one site that either does not have a
mismatch or is kinetically preferred out of
two possible cleavage sites.[58]

Nomenclature
Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia genus

co coli specific species

R RY13 strain

order of identification
I First identified
in the bacterium

Since their discovery in the 1970s, many

restriction enzymes have been identified;
for example, more than 3500 different
Type II restriction enzymes have been
characterized.[59] Each enzyme is named
after the bacterium from which it was
isolated, using a naming system based on
bacterial genus, species and strain.[60][61]
For example, the name of the EcoRI
restriction enzyme was derived as shown
in the box.

Applications
Isolated restriction enzymes are used to
manipulate DNA for different scientific
applications.
They are used to assist insertion of genes
into plasmid vectors during gene cloning
and protein production experiments. For
optimal use, plasmids that are commonly
used for gene cloning are modified to
include a short polylinker sequence (called
the multiple cloning site, or MCS) rich in
restriction enzyme recognition sequences.
This allows flexibility when inserting gene
fragments into the plasmid vector;
restriction sites contained naturally within
genes influence the choice of
endonuclease for digesting the DNA, since
it is necessary to avoid restriction of
wanted DNA while intentionally cutting the
ends of the DNA. To clone a gene
fragment into a vector, both plasmid DNA
and gene insert are typically cut with the
same restriction enzymes, and then glued
together with the assistance of an enzyme
known as a DNA ligase.[62][63]

Restriction enzymes can also be used to

distinguish gene alleles by specifically
recognizing single base changes in DNA
known as single-nucleotide
polymorphisms (SNPs).[64][65] This is
however only possible if a SNP alters the
restriction site present in the allele. In this
method, the restriction enzyme can be
used to genotype a DNA sample without
the need for expensive gene sequencing.
The sample is first digested with the
restriction enzyme to generate DNA
fragments, and then the different sized
fragments separated by gel
electrophoresis. In general, alleles with
correct restriction sites will generate two
visible bands of DNA on the gel, and those
with altered restriction sites will not be cut
and will generate only a single band. A
DNA map by restriction digest can also be
generated that can give the relative
positions of the genes.[66] The different
lengths of DNA generated by restriction
digest also produce a specific pattern of
bands after gel electrophoresis, and can
be used for DNA fingerprinting.
In a similar manner, restriction enzymes
are used to digest genomic DNA for gene
analysis by Southern blot. This technique
allows researchers to identify how many
copies (or paralogues) of a gene are
present in the genome of one individual, or
how many gene mutations
(polymorphisms) have occurred within a
population. The latter example is called
restriction fragment length polymorphism
(RFLP).[67]

Artificial restriction enzymes created by

linking the FokI DNA cleavage domain with
an array of DNA binding proteins or zinc
finger arrays, denoted zinc finger
nucleases (ZFN), are a powerful tool for
host genome editing due to their enhanced
sequence specificity. ZFN work in pairs,
their dimerization being mediated in-situ
through the FokI domain. Each zinc finger
array (ZFA) is capable of recognizing 9–12
base pairs, making for 18–24 for the pair.
A 5–7 bp spacer between the cleavage
sites further enhances the specificity of
ZFN, making them a safe and more
precise tool that can be applied in
humans. A recent Phase I clinical trial of
ZFN for the targeted abolition of the CCR5
co-receptor for HIV-1 has been
undertaken.[68]
Others have proposed using the bacteria
R-M system as a model for devising
human anti-viral gene or genomic vaccines
and therapies since the RM system serves
an innate defense-role in bacteria by
restricting tropism by bacteriophages.[69]
There is research on REases and ZFN that
can cleave the DNA of various human
viruses, including HSV-2, high-risk HPVs
and HIV-1, with the ultimate goal of
inducing target mutagenesis and
aberrations of human-infecting
viruses.[70][71][72] The human genome
already contains remnants of retroviral
genomes that have been inactivated and
harnessed for self-gain. Indeed, the
mechanisms for silencing active L1
genomic retroelements by the three prime
repair exonuclease 1 (TREX1) and excision
repair cross complementing 1(ERCC)
appear to mimic the action of RM-systems
in bacteria, and the non-homologous end-
joining (NHEJ) that follows the use of ZFN
without a repair template.[73][74]

Examples
Examples of restriction enzymes
include:[75]
 Recognition
Enzyme Source Cut
Sequence

5'---G AATTC--
5'GAATTC -3'
EcoRI Escherichia coli
3'CTTAAG 3'---CTTAA G--
-5'

5'--- CCWGG--
5'CCWGG -3'
EcoRII Escherichia coli
3'GGWCC 3'---GGWCC --
-5'

5'---G GATCC--
5'GGATCC -3'
BamHI Bacillus amyloliquefaciens
3'CCTAGG 3'---CCTAG G--
-5'

5'---A AGCTT--
5'AAGCTT -3'
HindIII Haemophilus influenzae
3'TTCGAA 3'---TTCGA A--
-5'

5'TCGA 5'---T CGA---3'

TaqI Thermus aquaticus
3'AGCT 3'---AGC T---5'

5'---GC GGCCGC--
5'GCGGCCGC -3'
NotI Nocardia otitidis
3'CGCCGGCG 3'---CGCCGG CG--
-5'

HinFI Haemophilus influenzae

5'GANTC 5'---G ANTC---3'
3'CTNAG 3'---CTNA G---5'
 5'--- GATC--
5'GATC -3'
Sau3AI Staphylococcus aureus
3'CTAG 3'---CTAG --
-5'

5'CAGCTG 5'---CAG CTG---3'

PvuII* Proteus vulgaris
3'GTCGAC 3'---GTC GAC---5'

5'CCCGGG 5'---CCC GGG---3'

SmaI* Serratia marcescens
3'GGGCCC 3'---GGG CCC---5'

5'GGCC 5'---GG CC---3'

HaeIII* Haemophilus aegyptius
3'CCGG 3'---CC GG---5'

5'GACGC 5'---NN NN---3'

HgaI[76] Haemophilus gallinarum
3'CTGCG 3'---NN NN---5'

5'AGCT 5'---AG CT---3'

AluI* Arthrobacter luteus
3'TCGA 3'---TC GA---5'

5'GATATC 5'---GAT ATC---3'

EcoRV* Escherichia coli
3'CTATAG 3'---CTA TAG---5'

5'---CAGCAGN25
5'CAGCAGN25NN NN---3'
EcoP15I Escherichia coli
3'GTCGTCN25NN 3'---GTCGTCN25NN
---5'

5'GGTACC 5'---GGTAC C---3'

KpnI[77] Klebsiella pneumoniae
3'CCATGG 3'---C CATGG---5'
 5'CTGCAG 5'---CTGCA G---3'
PstI[77] Providencia stuartii
3'GACGTC 3'---G ACGTC---5'

5'GAGCTC 5'---GAGCT C---3'

SacI[77] Streptomyces achromogenes
3'CTCGAG 3'---C TCGAG---5'

5'GTCGAC 5'---G TCGAC---3'

SalI[77] Streptomyces albus
3'CAGCTG 3'---CAGCT G---5'

5'AGTACT 5'---AGT ACT---3'

ScaI*[77] Streptomyces caespitosus
3'TCATGA 3'---TCA TGA---5'

5'ACTAGT 5'---A CTAGT---3'

SpeI Sphaerotilus natans
3'TGATCA 3'---TGATC A---5'

Streptomyces 5'GCATGC 5'---GCATG C---3'

SphI[77]
phaeochromogenes 3'CGTACG 3'---C GTACG---5'

5'AGGCCT 5'---AGG CCT---3'

StuI*[78][79] Streptomyces tubercidicus
3'TCCGGA 3'---TCC GGA---5'

5'TCTAGA 5'---T CTAGA---3'

XbaI[77] Xanthomonas badrii
3'AGATCT 3'---AGATC T---5'

Key:
* = blunt ends
N = C or G or T or A
W = A or T

See also
BglII – a restriction enzyme
EcoRI – a restriction enzyme
HindIII – a restriction enzyme
Homing endonuclease
List of homing endonuclease cutting
sites
List of restriction enzyme cutting sites
Molecular-weight size marker
REBASE (database)
Star activity
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External links
DNA Restriction Enzymes at the US
National Library of Medicine Medical
Subject Headings (MeSH)
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title=Restriction_enzyme&oldid=980867782"

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