RESTRICITION ENDONUCLEASES

SUBMITTED TO: SIR SAEED ULLAH KHATTAK

SUBMITTED BY: FATIMA HABIB
ROLL NO: 29
DATED: 20 JUNE 2019

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Contents
1.INTRODUCTION: ...................................................................................................................... 3
2.HISTORY .................................................................................................................................... 3
3.RECOGNITION SITES: ............................................................................................................. 3
4.NOMENCLATURE: ................................................................................................................... 4
5. TYPES: ....................................................................................................................................... 4
5.1 TYPE I RESTRICTION ENZYMES .................................................................................... 5
5.2 TYPE II RESTRICTION ENZYME..................................................................................... 5
5.3 TYPE III RESTRICTION ENZYME ................................................................................... 6
5.4 ARTIFICIAL RESTRICTION ENZYMES: ......................................................................... 7
5.4.1: ZINC-FINGER NUCLEASES ...................................................................................... 7
5.4.2 TALEN: .......................................................................................................................... 8
5.4.3 CRISPER CAS 9:(clustered regularly interspaced short palindromic repeats) . 8
6. MECHANISM OF ACTIOM OF RESTRICTION ENZYME: ................................................. 8
7. EXAMPLES: .............................................................................................................................. 9
8. APPLICATIONS: ..................................................................................................................... 10
REFERENCES: ............................................................................................................................ 12

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1.INTRODUCTION:
Restriction endonucleases are enzymes that cleave the sugar-phosphate backbone of
DNA strands. The vast majority of these enzymes have been isolated from bacteria,
where they carry out a host-defense function for the cell. These enzymes recognize a
specific DNA base sequence and cleave both strands of a double-stranded DNA
molecule at or near the recognition site. All restriction enzymes fall into one of three
classes, based upon their molecular structure and need for specific co-factors. Class I
endonucleases have a molecular weight around 300,000 Daltons, are composed of nonidentical
sub-units, and require Mg2+, ATP (adenosine triphosphate), and SAM (Sadenosyl-methionine) as
cofactors for activity. Class II enzymes are much smaller, with molecular weights in the range of
20,000 to 100,000 Daltons. They have identical subunits and require only Mg2+ as a cofactor [1].
The Class III enzyme is a large molecule, with a molecular weight of around 200,000 Daltons,
composed of non-identical subunits. These enzymes differ from enzymes of the other two classes
in that they require both Mg2+ and ATP but not SAM as co-factors. Class III endonucleases are
the rarest of the three types. [1]

2.HISTORY
Prior to 1968, the existence of restriction enzymes was unknown. However, the
phenomenon of restriction was well known. Restriction was the term given to the ability
of bacteria to recognize and attack any foreign DNA source whether it came from a virus
or from another strain of bacteria. In 1968, Matthew Meselson and Robert Yuan
reported that they had identified an enzyme in the bacterium Escherichia coli, strain K-
12, that appeared to be able to recognize and digest foreign DNAs [2]. This enzyme, they
concluded, could be the agent responsible for restriction. They coined the term
restriction endonuclease to refer to this enzyme. They further determined that such
enzymes would be ubiquitous among bacteria and that they would recognize and digest
any double-stranded DNA that was not protected by a specific pattern of DNA base
methylation [2]. Methylation of DNA involves adding a methyl-group (CH3) to the DNA
base such that the restriction enzyme will not recognize it.

3.RECOGNITION SITES:
Restriction enzymes recognize a specific sequence of nucleotides [3] 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 [4]. Many of them are palindromic, meaning the base sequence reads
the same backwards and forwards[5]. 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

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CATATG)[6]. 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 [7]. Different restriction enzymes that recognize the same sequence are known
as neo-schizomers. These often cleave in different locales of the sequence. Different enzymes that
recognize and cleave in the same location are known as iso-schizomers.

4.NOMENCLATURE:
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 [8]. Each
enzyme is named after the bacterium from which it was isolated, using a naming system based on
bacterial genus, species and strain [9] For example, the name of the Eco RI restriction enzyme
was derived as shown in the box.

Derivation of the Eco RI name [10]

Abbreviation Meaning Description

E Escherichia genus

co Coli specific species

R RY13 strain

order of identification
I First identified
in the bacterium

5. TYPES:
Based on the types of sequences identified, the nature of cuts made in the DNA, and the
enzyme structure, there are three classes.

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5.1 TYPE I RESTRICTION ENZYMES
 Type I restriction enzymes possess both restriction and modification activities. In this case,
the restriction will depend upon the methylation status of the target DNA sequence.
 Cleavage takes place nearly 1000 base pairs away from the restriction site [11].
 The structure of the recognition site is asymmetrical. It is composed of 2 parts. One part of
the recognition site is composed of 3-4 nucleotides while the other one contains 4-5
nucleotides. The two parts are separated by a non-specific spacer of about 6-8 nucleotides.
 For their function, the type I restriction enzymes require S- adenosylmethionine (SAM),
ATP, and Mg2+
 They are composed of 3 subunits, a specificity subunit which determines the recognition
site, a restriction subunit, and a modification subunit [12].

5.2 TYPE II RESTRICTION ENZYME

 Two separate enzymes mediate restriction and modification. Henceforth, DNA can be
cleaved in the absence of modifying enzymes. Although the target sequence identified by
the two enzymes is the same, they can be separately purified from each other [13].
 The nucleotides are cleaved at the restriction site only. The recognition sequence is
rotationally symmetrical, called palindromic sequence. The specific palindromic site can
either be continuous (e.g., KpnI identifies the sequence 5´-GGTACC-3´) or non-
continuous (e.g., BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any
nucleotide)
 These require Mg2+ as a cofactor but not ATP.
 They are required in genetic mapping and reconstruction of the DNA in vitro only because
they identify particular sites and cleave at those sites only [14].

How they work:

 The type II restriction enzymes first establish non-specific contact with DNA and bind to
them in the form of dimmers.
 The target sequence is then detected by a combination of two processes. Either the enzyme
diffuses linearly/ slides along the DNA sequence over short distances or hops/ jumps over
long distances.
 Once the target sequence is located, various conformational changes occur in the enzyme
as well as the DNA. These conformational changes, in turn, activate catalytic center.
 The phosphodiester bond is hydrolyzed, and the product is released.

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Structures of free, nonspecific, and specific DNA-bound forms of BamHI

5.3 TYPE III RESTRICTION ENZYME

 The type III enzymes recognize and methylate the same DNA sequence. However, they
cleave nearly 24-26 base pairs away [15].
 They are composed of two different subunits. The recognition and modification of DNA
are carried out by the first subunit- ‘M’ and the nuclease activity is rendered by the other
subunit ‘R’.
 DNA cleavage is aided by ATP as well as Mg2+ whereas SAM is responsible for stimulating
cleavage.

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  Only one of the DNA strand is cleaved. However, to break the double-stranded DNA, two
recognition sites in opposite directions are required [16].

5.4 ARTIFICIAL RESTRICTION ENZYMES:

In addition to the many natural restriction enzymes isolated from bacteria and archaea, it
is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence.
Examples:
5.4.1: ZINC-FINGER NUCLEASES
Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate
targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations
[17].

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 ZFN-Mediated Targeted Genome Editing
5.4.2 TALEN:
Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can
be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-
binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription
activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA
sequence, so when combined with a nuclease, DNA can be cut at specific locations.[1] The
restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in
situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger
nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.
5.4.3 CRISPER CAS9:(clustered regularly interspaced short palindromic repeats)
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a
guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR
sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known
as CRISPR-Cas9 that can be used to edit genes within organisms [20]. This editing process has a
wide variety of applications including basic biological research, development
of biotechnology products, and treatment of diseases.

6. MECHANISM OF ACTIOM OF RESTRICTION ENZYME:

The action of restriction enzymes is in many respects as varied as the enzymes themselves.
In general, however, the process is one of recognition of the binding site, binding of the enzyme
dimer to the DNA, cleavage of the DNA, and enzyme release Pingoud and Jeltsch note that this
scheme is a minimal scheme due to the complex variations in enzyme action that have been
observed [21]. To begin, all restriction endonucleases will bind DNA specifically and, with much
less strength, nonspecifically. This is a characteristic of many proteins that interact with DNA. It
is probable that even non-specific DNA binding will induce a conformational change in the
restriction enzyme dimer that will result in the protein adapting to the surface of the DNA strands
[22]. These changes are not the same as those that occur when the dimer binds to the recognition
site though. As the dimer slides along the DNA strands, it searches for recognition elements and,

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when these are encountered, an interaction between the protein and the DNA ensues in which the
non-specific complex is converted into a specific complex. This requires significant
conformational changes in both the protein and the DNA as well as expulsion of water molecules
from the protein/DNA interface so that more intimate contacts can be established [23]. In general,
intimate contact is held by 15 – 20 hydrogen bonds that form between the protein and the DNA
bases in the recognition site. These bonds are shown to be mediated through specific amino acids,
primarily ASP and GLU, held in a proper three-dimensional configuration. There are differences
among restriction enzymes with respect to how much water is expelled but, in all cases, it is a
substantially greater amount than is expelled during non-specific binding.

7. EXAMPLES:

Enzyme Source Recognition Sequence Cut

5'GAATTC 5'---G AATTC---3'

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

5'CCWGG 5'--- CCWGG---3'

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

5'GGATCC 5'---G GATCC---3'

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

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Enzyme Source Recognition Sequence Cut

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

8. 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 [25].
Restriction enzymes can also be used to distinguish gene alleles by
specifically recognizing single base changes in DNA known as single nucleotide
polymorphisms (SNPs) [26]. 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.

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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 [27]. 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) [28].
Artificial restriction enzymes created by linking the Fok I 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 [29].
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
[30]. 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.[68][69][70] 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 [31].

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