Restriction enzyme
A restriction enzyme or restriction endonuclease is an
enzyme that cleaves DNA into fragments at or near specific recognition sites
within the molecule known as restriction sites. These are also called Molecular
scissors that cut double stranded DNA molecules at specific points.
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.
The phenomenon was first identified in work done in the
laboratories of Salvador Luria, Weigle and Giuseppe Bertani in the early 1950s.
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.
Arbor and Dussoix in 1962 discovered that certain bacteria
contain Endonucleases which have the ability to cleave DNA.
In 1970 Smith and colleagues purified and characterized the
cleavage site of a Restriction Enzyme.
Werner Arbor, Hamilton Smith and Daniel Nathans shared the
1978 Nobel prize for Medicine and Physiology for their discovery of Restriction
Enzymes.
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.
Restrictions 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 defense mechanism against invading viruses.
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. Over 3000
restriction enzymes have been studied in detail, and more than 600 of these are available
commercially. These enzymes are routinely used for DNA modification in
laboratories, and they are a vital tool in molecular cloning.
Recognition site
Restriction enzymes recognize a specific sequence of
nucleotides 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
44 or 256bp, 6 bases, 46 or 4,096bp, and 8 bases would be
48 or 65,536bp.
] 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 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).
Inverted repeat palindromes are more common and have greater
biological importance than mirror-like palindromes.
EcoRI digestion produces "sticky" ends
whereas Sma1 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
Isoschizomers
Restriction enzymes that
have the same recognition sequence as well as the same cleavage site are known
as Isoschizomers
e.g.- Sma I
Neoschizomers
Restriction enzymes that have the same recognition sequence
but cleave the DNA at a different site within that sequence are Neoschizomers
e.g. Xma I
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.
They differ in their recognition sequence, subunit composition,
cleavage position, and cofactor requirements, as summarized 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 hydrolyses 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.
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 methylgroups 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.
Type
II
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.
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. These are
the most commonly available and used restriction enzymes. In the 1990s and
early 2000s, new
Type II site-specific deoxyribonuclease
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. These subgroups are defined using a letter suffix.
Type IIB restriction enzymes
(e.g., BcgI and BplI) are multimers, containing more than one subunit. 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. 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.
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. Type IIM restriction endonucleases, such
as DpnI, are able to recognize and cut methylated DNA. Type IIS restriction endonucleases
(e.g., FokI) cleave DNA at a defined distance from their
non-palindromic asymmetric recognition sites; 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
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.
These enzymes contain more than one subunit and require AdoMet
and ATP cofactors for their roles in DNA methylation and restriction digestion
, respectively.
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 and Mod. The Mod subunit recognizes 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 recognize 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).
Type
IV
Type IV enzymes recognize modified, typically methylated DNA and
are exemplified by the McrBC and Mrr systems of E. coli.
Action of McrBC,
a methyl-specific DNase. (A) Reaction in vitro. McrBC recognizes RmC (R = A or
G) and cleaves the DNA, usually near a recognition site. Cleavage requires two
recognition sites about 40–2000 bp (adapted from Raleigh91). (B) Restriction in
vivo. McrBC strongly restricts T-even phages whose DNA carries hydroxymethyl C
in place of C. However, it only weakly restricts plasmids and phages whose DNA
has been methylated by a modification enzyme
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.
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).
Such artificial restriction enzymes can target large DNA sites
(up to 36 bp) and can be engineered to bind to desired DNA sequences. Zinc finger nucleases are
the most commonly used artificial restriction enzymes and are generally used
in genetic engineering applications, but
can also be used for more standard gene cloning applications.
Other artificial restriction enzymes are based on the DNA binding domain
of TAL effectors.
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.
In 2017 a group in Illinois announced using an Argonaute protein taken from Pyrococcus furiosus (PfAgo)
along with guide DNA to edit DNA as artificial restriction enzymes.
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.
Nomenclature
|
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 |
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. 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.
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.
Restriction enzymes can also be used to distinguish gene alleles by specifically
recognizing single base changes in DNA known as single
nucleotide polymorphisms (SNPs). 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. 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).
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.
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. 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. 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.
Examples of restriction enzymes include:
|
Enzyme
|
Source
|
Recognition Sequence
|
Cut
|
|
XbaI
|
Xanthomonas badrii
|
5'TCTAGA
3'AGATCT
|
5'---T
CTAGA---3'
3'---AGATC T---5'
|
|
TaqI
|
Thermus aquaticus
|
5'TCGA
3'AGCT
|
5'---T CGA---3'
3'---AGC T---5'
|
|
StuI
|
Streptomyces
tubercidicus
|
5'AGGCCT
3'TCCGGA
|
5'---AGG CCT---3'
3'---TCC GGA---5'
|
|
SphI
|
Streptomyces
phaeochromogenes
|
5'GCATGC
3'CGTACG
|
5'---GCATG C---3'
3'---C
GTACG---5'
|
|
SpeI
|
Sphaerotilus natans
|
5'ACTAGT
3'TGATCA
|
5'---A
CTAGT---3'
3'---TGATC A---5'
|
|
SmaI*
|
Serratia marcescens
|
5'CCCGGG
3'GGGCCC
|
5'---CCC GGG---3'
3'---GGG CCC---5'
|
|
ScaI*
|
Streptomyces
caespitosus
|
5'AGTACT
3'TCATGA
|
5'---AGT ACT---3'
3'---TCA TGA---5'
|
|
Sau3AI
|
Staphylococcus
aureus
|
5'GATC
3'CTAG
|
5'--- GATC---3'
3'---CTAG ---5'
|
|
SalI
|
Streptomyces albus
|
5'GTCGAC
3'CAGCTG
|
5'---G
TCGAC---3'
3'---CAGCT G---5'
|
|
SacI
|
Streptomyces
achromogenes
|
5'GAGCTC
3'CTCGAG
|
5'---GAGCT C---3'
3'---C
TCGAG---5'
|
|
PvuII*
|
Proteus vulgaris
|
5'CAGCTG
3'GTCGAC
|
5'---CAG CTG---3'
3'---GTC GAC---5'
|
|
PstI
|
Providencia stuartii
|
5'CTGCAG
3'GACGTC
|
5'---CTGCA G---3'
3'---G
ACGTC---5'
|
|
NotI
|
Nocardia
otitidis
|
5'GCGGCCGC
3'CGCCGGCG
|
5'---GC GGCCGC---3'
3'---CGCCGG CG---5'
|
|
KpnI
|
Klebsiella
pneumoniae
|
5'GGTACC
3'CCATGG
|
5'---GGTAC C---3'
3'---C
CATGG---5'
|
|
HinFI
|
Haemophilus
influenzae
|
5'GANTC
3'CTNAG
|
5'---G ANTC---3'
3'---CTNA G---5'
|
|
HindIII
|
Haemophilus
influenzae
|
5'AAGCTT
3'TTCGAA
|
5'---A AGCTT---3'
3'---TTCGA A---5'
|
|
HgaI
|
Haemophilus
gallinarum
|
5'GACGC
3'CTGCG
|
5'---NN NN---3'
3'---NN NN---5'
|
|
HaeIII*
|
Haemophilus
aegyptius
|
5'GGCC
3'CCGG
|
5'---GG CC---3'
3'---CC GG---5'
|
|
EcoRV*
|
Escherichia coli
|
5'GATATC
3'CTATAG
|
5'---GAT ATC---3'
3'---CTA TAG---5'
|
|
EcoRII
|
Escherichia coli
|
5'CCWGG
3'GGWCC
|
5'--- CCWGG---3'
3'---GGWCC ---5'
|
|
EcoRI
|
Escherichia coli
|
5'GAATTC
3'CTTAAG
|
5'---G AATTC---3'
3'---CTTAA G---5'
|
|
EcoP15I
|
Escherichia coli
|
5'CAGCAGN25NN
3'GTCGTCN25NN
|
5'---CAGCAGN25 NN---3'
3'---GTCGTCN25NN ---5'
|
|
BamHI
|
Bacillus
amyloliquefaciens
|
5'GGATCC
3'CCTAGG
|
5'---G GATCC---3'
3'---CCTAG G---5'
|
|
AluI*
|
Arthrobacter luteus
|
5'AGCT
3'TCGA
|
5'---AG CT---3'
3'---TC GA---5'
|
Key:
* = blunt ends
N = C or G or T or A
W = A or T
* = blunt ends
N = C or G or T or A
W = A or T
Good work
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