Recombinant DNA Technology

D. K. Parihar
Asst. Professor
DEPARTMENT OF BIOTECHNOLOGY
Guru Ghasidas Vishwavidyalaya, Bilaspur-495009 (C.G.)
 Host-controlled restriction and modification
system

• Restriction systems allow bacteria to monitor the origin

of incoming DNA and to destroy it, if it is recognized as
foreign.

• Restriction endonucleases recognize specific

sequences in the incoming DNA (e.g. phages) and
cleave the DNA into fragments, either at specific sites or
more randomly, thus preventing it from successfully
replicating and parasitizing the cell (immunity system).
• The restrictive host must, of course, protect its own
DNA from the potentially lethal effects of the
endonuclease and so its DNA must be appropriately
modified.
• Modification involves methylation of certain bases at a
very limited number of sequences within DNA.
• Together, a restriction endonuclease and its ‘cognate’
modification methyl-transferase form a restriction-
modification system (R-M system)
EcoRI restriction endonuclease-methylase
system (type II)
 WHAT IS AN ENZYME?

• Enzymes are proteins and certain class of RNA

(ribozymes) which enhance the rate of a
thermodynamically feasible reaction and are not
permanently altered in the process.
 RESTRICTION ENZYMES

• A restriction enzyme (or restriction endonuclease) is an

enzyme that cuts double-stranded or single stranded
DNA at specific recognition nucleotide sequences
known as restriction sites.
• Restriction enzymes are molecular scissors
 HISTORY OF RESTRICTION ENZYME

• Rrestriction endonuclease of Escherichia coli K12 was purified by

Meselson and Yuan (1968).

• True restriction enzyme Hindll was isolated in 1970 by Kelly &

Smith 1970, Smith & Wilcox 1970 from Hemophilus influenzae.

• They also did subsequent discovery and characterization of

numerous restriction endonucleases.

• From then Over 3000 restriction enzymes have been studied in

detail, and more than 600 of these are available
commercially and are routinely used for DNA modification and
manipulation in laboratories
 HOW RESTRICTION ENZYMES WORKS?

• Restriction enzymes recognize a specific sequence of

nucleotides, and produce a double-stranded cut in the
DNA. these cuts are of two types:

• BLUNT ENDS.

• STICKY ENDS.
 BLUNT ENDS

• These blunt ended fragments can be joined to any other

DNA fragment with blunt ends.

• Enzymes useful for certain types of DNA cloning

experiments.
“STICKY ENDS” ARE USEFUL

DNA fragments with complimentary

sticky ends can be combined to
create new molecules which allows
the creation and manipulation of
DNA sequences from different
sources.
RECOGNITION SITE

• Recognition sequences vary widely , with lengths

between 4 and 8 nucleotides.
• Many of them are palindromic.
 PALINDROMES IN DNA SEQUENCES

• A palindromic sequence in DNA is one in which the 5’ to

3’ base pair sequence is identical on both strands (the 5’
and 3’ ends refers to the chemical structure of the DNA).
 NOMENCLATURE OF RESTRICTION ENZYME

• Each enzyme is named after the bacterium from

which it was isolated using a naming system based
on bacterial genus, species and strain.
For e.g EcoRI

Derivation of the EcoRI name

Abbreviation Meaning Description
E Escherichia Genus
co coli Species
R RY13 strain
order of identification
I First identified
in the bacterium
 TYPES OF RESTRICTION ENZYMES

• Restriction endonucleases are categorized into Four general

groups.
– Type I
– Type II
– Type III
– Type IIs

• These types are categorization based on:

 Their composition.
 Enzyme co-factor requirement.
 the nature of their target sequence.
 position of their DNA cleavage site relative to the target
sequence.
Characteristics of restriction endonucleases
Type I Type II Type III
 TYPE I - INTRODUCTION

• Type I restriction enzymes were the first to be identified

and are characteristic of two different strains (K-12 and
B) of E. coli.

• The recognition site is asymmetrical and is composed of

two portions – one containing 3-4 nucleotides, and
another containing 4-5 nucleotides – separated by a
spacer of about 6-8 nucleotides.
• Cutting site is 1,000bp away from recognition sequence
• Eg. EcoK, ECoB
 COFACTORS OF TYPE I

• Several enzyme cofactors include:

 S-Adenosyl methionine.

 Hydrolyzed adenosine triphosphate (ATP).

 Magnesium (Mg2+).
 SUBUNITS OF TYPE I

• Type I restriction enzymes possess three subunits:

o HsdR: is required for restriction.

o HsdM: necessary for adding methyl

groups to host DNA
(methyltransferase activity).

o HsdS: important for specificity of cut site

recognition in addition to its
methyltransferase activity.
• The recognition sequences are quite long with
no recognizable features such as symmetry.
• The enzyme cuts unmodified DNA at some
distance from the recognition sequence.
• However, because the methylation reaction is
performed by the same enzyme which mediates
cleavage, the target DNA may be modified
before it is cut.
• These enzymes read the methylation status of their
recognition sequence, compare the methylation status of
two adenines within the recognition sequence, and if
both adenines are un-methylated (a signal that the DNA
is non-host DNA), the enzyme undergoes a
conformational switch that turns the enzyme into a
molecular motor and endonuclease.
 TYPE III

• Type III restriction enzymes ) 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
 Type IV

• Modified DNA (methylated, hydroxymethylated and

glucosyl-hydroxymethylated bases).
• Recognition sequences have not been well defined
• Examples: McrBC and Mrr systems of E. coli.
 ARTIFICIAL RESTRICTION ENZYMES

• Generated by fusing a natural or engineered DNA

binding domain to a nuclease domain

• can target large DNA sites (up to 36 bp)

• can be engineered to bind to desired DNA sequences

 TYPE II

• These are the most commonly available and used

restriction enzymes

• They are composed of only one subunit.

• Their recognition sites are usually undivided and

palindromic and 4-8 nucleotides in length, they recognize
and cleave DNA at the same site.

• They do not use ATP or AdoMet for their activity – they

usually require only Mg2+ as a cofactor.
 CUTS OF TYPE II

• Type II restriction enzymes can generate two different

types of cuts depending on whether they cut both
strands at the centre of the recognition sequence:

• The former cut will generate “blunt ends” with no

nucleotide overhangs.

• The latter, generates “sticky” or “cohesive” ends

• Most, but not all, type II restriction endonucleases
recognize and cleave DNA within particular sequences of
4 - 8 nucleotides.
• Sequence have two fold axis of rotational symmetry.
Palindromes.
– For example, the restriction and modification
enzymes [Link] and [Link] recognize the
sequence:
 APPLICATION OF RESTRICTION ENZYMES

• They are used in gene cloning and protein expression

experiments.

• Restriction enzymes are used in biotechnology to cut

DNA into smaller strands in order to study fragment
length differences among individuals (Restriction
Fragment Length Polymorphism – RFLP).

• Each of these methods depends on the use of agarose

gel electrophoresis for separation of the DNA fragments.
• The position at which the restricting enzyme cuts is
shown by the symbol “/”.
• The nucleotides methylated by the modification enzyme
are marked with an asterisk.
– For example EcoRI
5′-G/AA*T T C-3′
3′-C T T A*A/G-5′
• Only one strand of DNA is represented in practice,
which runs in the 5′ to 3′ direction.
– Thus the EcoRI recognition sequence would be shown as:
G/AATTC.
• EcoRI makes single-stranded breaks four bases apart in the
opposite strands of its target sequence so generating
fragments with protruding 5′ termini:

5′-G 5′-AATTC-3′
3′-CTTAA-5′ G-5′

• Not all type II enzymes cleave their target sites like EcoRI.
Some, such produce fragments bearing 3′ overhangs,
– Example PstI
CTGCA/G

• Some RE, such as SmaI (CCC/GGG), produce blunt or flush

ends.
Sticky End cut:

HindIII

EcoRI

Blunt End cut:

AluI

HaeIII
 ISOSCHIZOMER

• Restriction enzymes specific to the same recognition

sequence.
For example:

SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each

other.
 Neoschizomer

• Enzyme that recognizes the same sequence but cuts it

differently is a neoschizomer.

For example:

SmaI (CCC/GGG) and XmaI (C/CCGGG) are neoschizomers of

each other.
Frequency
• In DNA with 50% G+C content and a random distribution
of the 4-bases,
– A 4-base recognition site occurs every 44 (256) bp.
– A 6-base recognition site occurs every 46 (4096) bp
– An 8-base recognition sequence every 48 (65,536) bp.
• Not a random distribution of the four bases and many organisms
can be AT- or GC-rich, e.g. the nuclear genome of mammals is
40% G+C and the dinucleotide CG is fivefold less common than
statistically expected.
• Similarly, CCG and CGG are the rarest trinucleotides in most A+T-
rich bacterial genomes and CTAG is the rarest tetranucleotide in
G+C-rich bacterial genomes.
• Thus different restriction endonucleases with six-base recognition
sites can produce average fragment sizes significantly different
from the expected 4096 bp.
Preference of cleavage

• Certain RE show preferential cleavage of some sites in

the same DNA molecule.
– For example, phage λ DNA has five sites for EcoRI but the
different sites are cleaved non-randomly
– The site nearest the right terminus is cleaved 10 times faster
than the sites in the middle of the molecule.
• There are four sites for SacII in λ DNA but the three sites
in the center of the molecule are cleaved 50 times faster
than the remaining site.
Preference of cleavage

• NarI, NaeI, and SacII require simultaneous interaction

with two copies of their recognition sequence before they
will cleave DNA.

• Thus NarI will rapidly cleave two of the four recognition

sites on plasmid pBR322 DNA but will seldom cleave the
remaining two sites.
DNA Ligase
 DNA ligase – joining DNA molecules

• DNA ligase (polydeoxyribonucleotide synthase) is an

important cellular enzyme, as its function is to repair
broken phosphodiester bonds that may occur at random
or as a consequence of DNA replication or
recombination.

• DNA ligase catalyses the formation of phosphodiester bond

between the 5’-phosphate of one strand of DNA or RNA and
the 3’-hydroxyl of another.

• This enzyme is used to covalently link or ligate fragments of

DNA together
• Ligase is like a molecular stitcher. They are ubiquitous
and essential for all cells and perhaps at all times.
• DNA ligase is the enzyme that joins two single stranded
DNA fragments by catalyzing the formation of an inter-
nucleotide ester bond between phosphate and
Deoxyribose.
• It is active during DNA replication, DNA repair and
DNA recombination.
 There are two forms of DNA ligase

1. NAD requiring: NAD (+) dependent DNA ligases are

present in bacteria, some Entomopox viruses and Mimi
virus.
2. ATP requiring: ATP-dependent DNA ligases are
ubiquitous.
• ligates break in one of the double stranded DNAby
phosphodiester bond formation
• Use the free 5’p of a nucleotide of one segment and the
free 3’OH group of the neighboring segment,
• They catalyze the reaction to form a covalent bond, thus
they seal the nick.
• For the enzyme to function it requires ds DNA with such
gaps or nicks in one of the strands.
• The nick should not contain any missing nucleotide in
between the ends.
• A minimal length of the ds strand DNA needed for its
function is at least 4 to 8 base pair length.
 Reaction mechanism of bacterial DNA ligase
1. In first step the reversible adenylation of ligase with NAD+ as the adenylyl donor
takes place.
2. Ligase then transfers the AMP to the 5′ phosphate end of nicked duplex DNA
3. finally catalyzes nick closure and the release of AMP.
 Reaction

• Enzyme + ATP-> Enzyme-Amp + Ppi

• Enzyme binds to broken region and adds

Phosphate group to 5’end P,

• Then brings ligation between 3’OH and

Phosphate to release AMP
 • Many DNA Ligases are identified from different sources,
which have their own specific characters, molecular
weights, requirements and mode of functions.

[Link] DNA Ligase T4 DNA Ligase T7 DNA Ligase

[Link] (kd) 75 60 41
Sub units Monomer Monomer Monomer
Cofactors required NAD ATP ATP
Catalyze covalent bond formation Yes Yes Yes
between 3’OH and 5’p of nucleotides
Perform sticky end ligation Yes, efficient Yes efficient Yes, efficient
Perform blunt end ligation No Yes, not efficient Yes,
Blunt or sticky ends, require 5’phosphate Absolutely required Absolutely Absolutely
group and 3’OH group required required
Can they use 2’OH groups No No No
Can they join ssDNA end to end No No No
Can they ligate RNA strands on a DNA No Yes ?
strand
Can they ligate DNA strands on a RNA No Yes ?
strand
• DNA ligases play an integral role in DNA repair
and replication through catalyzing the formation
of phosphodiester bonds.
• Two types of DNA ligase have been identified:
– ATP-dependent DNA ligases (EC [Link]), and
– NAD+-dependent DNA ligases (EC [Link]).
 The ATP-dependent DNA ligases are divided into four
classes

– DNA ligase I:- links Okazaki fragments to form a

continuous strand of DNA
– DNA ligase II:- is an alternatively spliced form of DNA
ligase III:- is found only in non-dividing cells
– DNA ligase III :- is involved in base excision repair
– DNA ligase IV:- is involved in the repair of DNA double-
strand breaks by non-homologous end joining (NHEJ).
 Eukaryotic DNA Ligases
• Structure, mode of function and mechanism of catalysis of
Eukaryotic ligases is same as that of prokaryotic Ligases.
• Two types of enzymes are present; both are located in the
nucleus.
• Type I is mostly involved in replication and found in large
numbers in proliferating cells or during cell division.
• DNA-ligase type II was found to be very active during DNA
repair or recombination events.
• The enzymes are heat labile.
A List of Eukaryotic Ligases

Properties Ligase-1 Ligase-2

Molecular mass (kd) 85-125 68
ATPs requirement Yes, require 2ATPs Yes, require 4 ATPs
Blunt end ligation Yes No
Sticky end ligation Yes Yes
Location Nucleus Nucleus
Fractional activity 90% <10%
Overall function Replication Repair

Eukaryotic Ligases (Mammalian)

Name [Link] Subunits Cofactor

Ligase-I 85-125 Monomer ATP
Ligase-II 68 Monomer ATP
 RNA Ligase

• RNA Ligases are also located in nuclei and their activity

is high when transcriptional activity is at its peak.
• In bacterial hosts, T4 Phage produces an RNA ligase 43
KD (gp 63).
• RNA Ligases extracted from wheat germ and yeast cells
do exhibit different activities.
• They join 3’ OH of one piece of RNA to 5’P of another
piece of RNA in 5’ to 3’ end-to-end manner.
 RNA Ligase

• Enzymes require first two to three nucleotides of the

RNA fragments to recognize the ends and bind to
perform catalytic activity.
• Most of the RNA ligases found in eukaryotic systems are
involved in RNA splicing.
• They can also phosphorylate 5’ends and can cleave
cAMP leaving Phosphate group at 2’end.
 Bacteriophage T4 DNA Ligase (ATP)

• The most widely used DNA ligase is derived from the T4

bacteriophage.
• It is a monomeric polypeptide
• MW 68KDa is encoded by bacteriophage gene30.
• It has broder specificity and repairs single strended
Nicks in duplex DNA, RNA or DNA:RNA hybrids.
 Application

1. ligation of cohesive ends:

2. ligation of blunt ended termini: this reaction is much
slower than ligation of sticky ends and the ligation is
improved by addition of monovalent cation and low
concentration of PEG
3. Ligation of synthetic linkers or adapter
 [Link] DNA ligase

• It is derived from [Link] cell and requires NAD+ as

cofacter.
• It is a monomeric enzyme of MW 74KDa which
catalyzes the formation of the phosphodiester bond in
duplex DNA containing cohesive ends.
• This enzyme has narrower substrate specificity, making
it a useful tool in specific application.