This document discusses restriction enzymes, which are important tools in genetic engineering and recombinant DNA technology. Restriction enzymes cut DNA at specific recognition sequences and are used to cut DNA into fragments that can then be recombined in new ways. The document provides details on the discovery and functions of various restriction enzymes as well as other enzymes used in genetic engineering such as DNA ligase, alkaline phosphatase, polynucleotide kinase, and reverse transcriptase. It also discusses the use of restriction enzymes to build the first synthetic bacterial genome.

1. Genetic Engineering
Chapter 2- Restriction Enzymes
Hikmet Geçkil, Professor
Department of Molecular Biology and Genetics
Inonu University

2. Restriction Enzymes
• The most important tools in the recombinant
DNA technology toolbox.
• Bacteria and archaea have evolved mechanisms
to protect themselves from viral infections.
• Restriction endonucleases are part of the natural
defence mechanisms of bacteria against
incoming foreign DNA.
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3. • In early 1950s, Salvador Luria showed that some
strains of bacteria were more resistant to
bacteriophages than others.
• In the 1960s, Werner Arber and Stuart Linn
observed that bacteriophage DNA was degraded
and cut into pieces in resistant strains of bacteria.
• In 1970, Hamilton Smith successfully purified a restriction enzyme
(HindII) from bacterium, Haemophilus influenzae showing that the
enzyme cut bacteriophages DNA in the center of a specific six-base-
pair sequence, while it did not cut at this very same DNA sequence
when it occurred in host cell DNA (i.e., H. influenzae DNA).
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4. • In 1971 Dan Nathans showed different
combinations of restriction enzymes could cut
SV40 genome into specific sizes which could
be used to deduce a physical map of the SV40
genome (restriction mapping).
• For this groundbreaking set of discoveries,
Arber, Smith, and Nathans were jointly
awarded the Nobel Prize in Physiology or
Medicine in 1978.
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5. • To date, more than 4,000 different restriction
enzymes have been isolated from a wide
variety of bacterial strains.
• The recognition sequences of these enzymes
are typically four to six base pairs in length,
and they are usually palindromic, (i.e, their
recognition sequence reads same in the 5' to
3' direction on both DNA strands.
5'- G A A T T C -3'
3'- C T T A A G -5'
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6. • Most enzymes have recognition sites of 4 or 6
nucleotides.
• Thus, a particular four-nucleotide motif would be
expected to occur on average once every 44 (i.e.
256) nucleotides.
• So the average length of fragments generated by
enzymes with such sites would be 256 base-pairs.
• In practice, that does not happen as the bases do
not occur with equal frequencies.
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7. • Different REs can recognize the same
sequence (they are said to be isoschizomers)
• However, they do not necessarily cut at the
same position within the recognition site
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8. Restriction Enzymes Require Magnesium for
Catalytic Activity
• REs as well as many other enzymes that act on phosphate-
containing substrates require Mg2+ or some other similar
divalent cation for activity.
• The magnesium ion was found to be bound to six ligands:
three are water molecules, two are carboxylates of the
enzyme's aspartate residues, and one is an oxygen atom of
the phosphoryl group at the site of cleavage.
• The magnesium ion holds a water molecule in a position
from which the water molecule can attack the phosphoryl
group and, in conjunction with the aspartate residues,
helps polarize the water molecule toward deprotonation.
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9. Five different categories of restriction enzymes
1. Type I restriction enzymes cut DNA at random locations far from
their recognition sequence
2. Type II cut within or close to their recognition sequence (the most
useful ones in Rec DNA experiments)
3. Type III cut outside of their recognition sequence
4. Type IV typically recognize a modified recognition sequence
5. 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. Promising genome
editing tools for future applications as we will see later in this
lecture.
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10. Genetic Engineering/Hikmet
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Chapter 2: Restriction Enzymes 10
DNA fragments can be cut
using restriction enzymes and
recombined using DNA ligase.
Upper panel: the restriction
enzyme HindII cuts DNA into
fragments with blunt ends. The
restriction enzyme EcoR1, by
contrast, cuts DNA into
fragments with sticky ends.
Lower panel: DNA molecules
cut with EcoR1 have
complementary sticky ends that
pair when the DNA fragments
are mixed together.

11. • In 2010, forty years after he purified the first
restriction enzyme, Smith was part of the
research team that used these very enzymes
to build the first synthetic bacterial cell.
• Led by Craig Venter, they synthesized the one
million base-pair Mycoplasma mycoides
bacterial genome.
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12. How do host REs manage to degrade viral DNA while
sparing their own? Restriction Modification System
• Protection by Methylation
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The recognition
sequence for EcoRV
endonuclease (left) and
the sites of methylation
(right) in DNA protected
from the catalytic action
of the enzyme. The host
DNA is protected by
other enzymes called
methylases, which
methylate adenine bases
within host recognition
sequences.
For each restriction endonuclease, the
host cell produces a corresponding
methylase that marks the host DNA and
prevents its degradation. These pairs of
enzymes are referred to as restriction-
modification systems.

13. • The reaction catalyzed by restriction endonucleases is the
hydrolysis of the phosphodiester backbone of DNA.
• Specifically, the bond between the 3′ oxygen atom and the
phosphorus atom is broken.
• The products of this reaction are DNA strands with a free
3′-hydroxyl group and a 5′-phosphoryl group.
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Hydrolysis of a Phosphodiester Bond
All restriction enzymes catalyze the hydrolysis of DNA phosphodiester bonds,
leaving a phosphoryl group attached to the 5′ end. The bond that is cleaved is
shown in red.

14. Other nucleases
• Four other useful
nucleases used in
genetic engineering:
1. Bal 31
2. Exonuclease III
3. DNase I
4. S1-nuclease
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15. • Taq polymerase is a thermostable DNA polymerase
isolated in 1976 from the thermophilic bacterium Thermus
aquaticus that lives in hot springs and hydrothermal vents.
• It is frequently used in polymerase chain reaction (PCR), a
method for greatly amplifying short segments of DNA.
• Taq's optimum temperature for activity is 75–80°C, with
a half-life of greater than 2 hours at 92°C, 40 minutes at
95°C and 9 minutes at 97°C, and can replicate a 1000 base
pair strand of DNA in less than 10 seconds at 72°C.
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Thermostable DNA Polymerases

16. • Taq makes DNA products that have A
(adenine) overhangs at their 3' ends. This may
be useful in TA cloning, whereby a cloning
vector (such as a plasmid) that has a T
(thymine) 3' overhang is used, which
complements with the A overhang of the PCR
product, thus enabling ligation of the PCR
product into the plasmid vector.
• One of Taq's drawbacks is its lack
of 3' to 5' exonuclease proofreading activity
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17. • Archaeal Pfu DNA polymerase has superior
thermostability and proofreading properties
compared to Taq DNA polymerase.
• Unlike Taq, Pfu DNA polymerase possesses 3' to
5' exonucleaseproofreading activity, meaning that
as the DNA is assembled from the 5' end to 3'
end, the exonuclease activity immediately
removes nucleotides misincorporated at the 3'
end of the growing DNA strand.
• Consequently, Pfu DNA polymerase-generated
PCR fragments will have fewer errors than Taq-
generated PCR inserts.
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18. • Vent polymerase is a thermostable DNA
polymerase used for the polymerase chain
reaction. It was isolated from Thermococcus
litoralis.
• Vent DNA Polymerase is a high-fidelity
thermophilic DNA polymerase. The fidelity of
Vent DNA Polymerase is 5-15-fold higher than
that observed for Taq DNA Polymerase.
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19. Enzymes that modify the ends of DNA molecules
• The enzymes alkaline phosphatase,
polynucleotide kinase, and terminal
transferase act on the termini of DNA
molecules and provide important functions
that are used in a variety of ways.
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20. • Two primary uses for alkaline phosphatase
1. Removing 5' phosphates from plasmid and
bacteriophage vectors that have been cut with
a restriction enzyme. In subsequent ligation
reactions, this treatment prevents self-ligation
of the vector and thereby greatly
facilitates ligation of other DNA fragments into
the vector (e.g. subcloning).
2. Removing 5' phosphates from fragments of
DNA prior to labeling with radioactive
phosphate. Polynucleotide kinase is much
more effective in phosphorylating DNA if the
5' phosphate has previously been removed.
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21. • Polynucleotide kinase (PNK) adds a phosphate group to DNA and RNA.
• It is widely used in molecular biology and genetic engineering to add
radio-labelled (e.g., 32P) phosphates.
• The enzymatic activity of PNK is utilized in two types of reactions:
– In the "forward reaction", PNK transfers the phosphate from ATP to the 5' end of a
polynucleotide (DNA or RNA) which lacks a 5' phosphate.
– In the "exchange reaction", target DNA or RNA that has a 5' phosphate is incubated with
ADP and the enzyme transfer sthe phosphate from the nucleic acid onto an ADP, forming
ATP and thus leaves a dephosphorylated target. PNK will then perform a forward
reaction and transfer a phosphate from ATP onto the target nucleic acid.
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22. • Terminal deoxynucleotide transferase adds
nucleotides at 3' -OH end (like Klenow
fragment) but does not require any
complementary sequence and does not copy
any DNA sequence (unlike Klenow fragment).
• The enzyme adds nucleotide whatever comes
into its active site and it does not show any
preference for any nucleotide.
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23. DNA ligase – joining DNA molecules
• DNA ligase 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.
• In genetic engineering it is used to seal
discontinuities in the sugar—phosphate chains
that arise when recombinant DNA is made by
joining DNA molecules from different sources.
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24. Other enzymes
• The Klenow fragment
• Reverse transcriptase
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25. • The Klenow fragment, reported in 1970, is a
large protein fragment produced when DNA polymerase
I from E. coli is enzymatically cleaved by
the protease subtilisin. It has 5→3 polymerase and 3→5
exonuclease activity, but no 5→3 exonuclease function.
• The Klenow fragment is used in:
– Synthesis of double-stranded DNA from single-stranded
templates
– Filling in receded 3' ends of DNA fragments to make 5' overhang
blunt
– Digesting away protruding 3' overhangs
– Preparation of radioactive DNA probes
• The Klenow fragment was he original enzyme used for
PCR, before replaced by the Taq polymerase.
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26. • Reverse transcriptase is an RNA-dependent
DNA polymerase, and therefore produces a
DNA strand from an RNA template.
• It has no associated exonuclease activity.
• The enzyme is used mainly for copying mRNA
molecules in the preparation of cDNA
(complementary or copy DNA) for cloning.
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