Lecture 2

Enzymes in rDNA technology

August 7, 2018
 Enzymes in rDNA Technology:
Nucleases
• Nucleases degrade DNA molecules.
• They break the phosphodiester bonds that link
one nucleotide to the next in a DNA strand.
• There are two different kinds of nuclease:
• 1) Exonucleases: these remove nucleotides one
at a time from the end of a DNA molecule.
• 2) Endonucleases: these are able to break
internal phosphodiester bonds within a DNA
molecule.
• The differences in exonucleases lies in the number of
strands that are degraded when a double-stranded
molecule is attacked.
• The enzyme called Bal31 (purified from the bacterium
Alteromonas espejiana) is an example of an exonuclease.
• It removes nucleotides from both strands of a double-
stranded molecule.
• The greater the length of time that Bal31 is allowed to
act on a group of DNA molecules, the shorter the
resulting DNA fragments will be.
 Nucleases
• E. coli exonuclease III degrades just one strand
of a double-stranded molecule.
• This leaves single-stranded DNA as the
product.
 DNA-degrading enzymes: Endonucleases

• Examples:
• 1) S1 endonuclease.
• This can be isolated from Aspergillus oryzae a
fungus.
• It only cleaves single strands.
• One example of its use is in intron mapping in
a technique called S1 mapping.
• 2) Deoxyribonuclease I (DNase I).
• This can be prepared from cow pancreas.
• It cuts both single and double-stranded
molecules.
• DNase I is non-specific in terms of how and
where it cuts DNA.
• It can cut DNA at any internal phosphodiester
bond.
• If DNA is exposed to DNAse for long, a mixture
of mononucleotides and very short
oligonucleotides are obtained.
• It differs from restriction endonucleases which
cleave double-stranded DNA only at a limited
number of specific recognition sites.
• One use of DNAse is to locate regions of DNA-
protein interaction sites in regions where
regulatory sequences of genes are located.
• This technique is called DNAse foot printing.
 Type II Restriction endonucleases
• Restriction enzymes are used in rDNA
technologies to cut DNA into sizeable pieces.
• These are proteins and enzymes as such.
• They have the same characteristics of
enzymes including acting at specific
temperatures and requirement for specific
buffers.
• The enzymes are found in bacteria.
 Restriction enzymes
• In bacteria REs are used to defend bacteria by
cutting foreign DNA from viruses.
• Since they are from bacteria, they are named
after the genera and species from which they
come.
• EcoR1 stands for Escherichia coli Restriction
Endoclease 1.
• This enzyme was isolated from that bacterium.
• The central feature of type II restriction
endonucleases (restriction endonucleases) is
that each enzyme has a specific recognition
sequence in a given DNA molecule.
• It cuts a DNA molecule at the recognition
sequence and nowhere else.
• For example, the restriction endonuclease
called PvuI (isolated from Proteus vulgaris) cuts
DNA only at the hexanucleotide CGATCG.
• In contrast, a second enzyme from the same
bacterium, called PvuII, cuts at a different
hexanucleotide, in this case CAGCTG.
• Many recognize hexanucleotide target sites.
• Others cut at four, five, eight, or even longer
nucleotide sequences.
 Type II REs
• Sau3A (from Staphylococcus
• aureus strain 3A) recognizes GATC.
• AluI (Arthrobacter luteus) cuts at AGCT.
• There are also examples of restriction
endonucleases with degenerate recognition
sequences.
 1 gcgcggagcg cgatcgccctccgttcc ggtggggggt ggcaactggc aacgcaaaag
61 cctgactcca atcccattgc tgctggaaag cacaaagcag cgatggtaaa agaggatgaa
121 ggccatggcg acagagtggc cgccgacgct cctttccgtg tggtgtaggg ctgcagattc
181 cttcgcgctc ctcctgctcc cagcgaggaa aagagagcac gaactaaact aggaaccatc
241 cctccccggc attcattccc ttgccctttt ttcaccgaat ggattcggtt gttgctttcc [BamHI]
301 tgttcaggtg ccttgttctt ggcctccttc gcacgcaaag aaccgccggt cgtattgctc
361 tccgcactca cccaccgcgc tgctgctgct cgactgctcg tggaagggtt cttattggcc
421 gcctgttcct gaattccggt ttcttgctct gcttttgagt ggagccgctg tggagtggac [EcoRI]
481 ggtgtggtgt ggtgtgggtc ggcggtgagg tgaaaggctt ggtttctttc tttcttgcga
541 gggaggataa gggaaaggga aggcgaagag gaggatggcc ggtaccgggg cggcgccgc
[KpnI]
601 gccgaagcag gaggagctgc agccgcaccc ggtgaaggac cagcttccca gcgtgtccta
661 ctgcatcaca agcccaccgc cctggcctga ggctgtcatc cttggattcc agcactacat
• This means that they cut DNA at any one of a
family of related sites.
• HinfI (Haemophilus influenzae strain Rf), for
instance, recognizes GANTC.
• It cuts at GAATC, GATTC, GAGTC, and GACTC.
 Blunt and Overhang Cutters
• The exact nature of the cut produced by a
restriction endonuclease is important in the
design of a gene cloning experiment.
• Many restriction endonucleases cut DNA in
the middle of the recognition sequence.
• The DNA produced has blunt or flush ends.
• Examples of enzymes that leave blunt ended
DNA are EcoRV, PvuII and AluI.
• Other restriction endonucleases cut DNA in a
slightly different way.
• With these enzymes the two DNA strands are not
cut at exactly the same position.
• Instead the cleavage is staggered usually by two
or four nucleotides.
• The resulting DNA fragments have short single-
stranded overhangs at each end.
• These are called sticky or cohesive ends,
• The base pairing between them can stick the
DNA molecule back together again.
• One important feature of sticky end enzymes
is that restriction endonucleases with different
recognition sequences may produce the same
sticky ends.
• BamHI (recognition sequence GGATCC) and
BglII (AGATCT) are examples of REs both
produce GATC sticky ends.
• The same sticky end is also produced by Sau3A,
which recognizes only the tetranucleotide GATC.
• Fragments of DNA produced by cleavage with
either of these enzymes can be joined to each
other as each fragment carries a
complementary sticky end.
• Enzymes that recognize similar DNA sequences
are called isoschizomers.
• RE type II also show rotational symmetry for
target sites.
• They cut at specific temperatures e.g. 25C, and
37C.
• They are names using the first letter of the genus
name and the first tow letters of the species
name.
• E.g. EcoRI for Escherichia coli restriction enzyme I
• Some are methylation sensitive meaning that
where DNA is methylated they may not cut.
 DNA Ligases

• In the cell the function of DNA ligase is to

repair single-stranded breaks.
• These are sometimes called discontinuities.
• They arise in double-stranded DNA molecules
during, for example, DNA replication.
• DNA ligases from most organisms can also join
together two individual fragments of double-
stranded DNA
• T4 DNA ligase is used to make recombinant
DNA molecules in test tubes.
• This is the enzyme traditionally used for
construction of recombinant DNA molecules in
labs.
 Polymerases
• DNA polymerases are enzymes that synthesize a
new strand of DNA.
• They use nucleotide sequences in an existing DNA
or RNA to make a complementary sequence.
• The nucleotide used is called a template molecule.
• Most polymerases can function only
• if the template possesses a double-stranded
region that acts as a primer for initiation of
polymerization.
• Four types of DNA polymerase are used
routinely in genetic engineering.
• The first is DNA polymerase I.
• This enzyme is usually prepared from E. coli.
• It attaches to a short single-stranded region
within a double stranded DNA molecule.
• This is sometimes called a nick.
• It then synthesizes a completely new strand,
degrading the existing strand as it proceeds.
• DNA polymerase I is has a dual activity.
• It can synthesize/polymerize DNA by its DNA
polymerization activity.
• It can also break down/degrade DNA by its
DNA degradation/nuclease/hydrolytic activity.
• The polymerase and nuclease activities of
DNA polymerase I are controlled by different
parts of the enzyme molecule.
• The nuclease activity is contained in the first
323 amino acids of the polypeptide.
• If these first 323 amino acids are removed, a
modified enzyme remains.
• This enzyme retains the polymerase function.
 Klenow polymerase
• Its nuclease activity is lost.
• This enzyme is unable to degrade DNA.
• This modified enzyme is called the Klenow
fragment.
• It can still use a template ssDNA to make a
complementary DNA strand.
• Klenow has no nuclease activity.
• It cannot continue synthesis once the nick is
filled.
• Several other enzymes—natural polymerases
and modified versions—have similar
properties to the Klenow fragment.
• The major application of these polymerases is
in DNA sequencing.
• The second is Taq DNA polymerase.
• This is used in the polymerase chain reaction (PCR).
• It is isolated from the bacterium Thermus
aquaticus.
• This organism lives in hot springs.
• Many of its enzymes, including the Taq DNA
polymerase, are thermostable.
• They are resistant to denaturation by heat
treatment.
• This is the special feature of Taq DNA
polymerase that makes it suitable for PCR,
because.
• If it was not thermostable it would be
inactivated when the temperature of the
reaction is raised to 94°C to denature the
DNA.
• The final type of DNA polymerase that is
important in genetic engineering is reverse
transcriptase (RT).
• RTs are enzymes involved in the replication of
several kinds of virus.
• Examples are the HIV RT, Avian myeloblastosis
virus (AMV) RT and Moloney’s murine
leukemia virus (MMLV) RT.
• An RT uses RNA and not DNA as a template.
• It makes a complementary strand of the RNA
to make DNA.
• This property is used to make called
complementary DNA (cDNA) cloning.
• This can be used to screen gene expression in
transgenic organisms.
 Enzymes that modify DNA
• These enzymes modify DNA molecules by addition
or removal of specific chemical groups e.g
phosphate, sugar etc.
• Examples are:
• Alkaline phosphatase:
• This can be extracted from E. coli, calf intestinal
tissue, or arctic shrimp.
• As the name says, it removes the phosphate group
present at the 5′ terminus of a DNA molecule.
• Polynucleotide kinase:
• This is extracted from E. coli infected with T4
phage.
• It catalyzes the reaction which is the reverse of
that catalyzed by alkaline phosphatase.
• It adds the phosphate groups onto free 5′
termini of a DNA molecule.
• Terminal deoxynucleotidyl transferase
• This is extracted from calf thymus tissue.
• It adds one or more deoxyribonucleotides
onto the 3′ terminus of a DNA molecule.
• It can be used in the insertion of blunt-ended
DNA molecules into vectors also cut with blunt
end cutting enzymes such as EcoRV.
• A more sophisticated, but easier and generally
more efficient way of carrying out blunt end
ligation, is to use a special type of enzyme
called a DNA topoisomerase.
• DNA topoisomerases remove or add turns
from the double helix or to a double-stranded
DNA molecule.
• Turns are removed during DNA replication in
order to unwind the helix.
• This enables each polynucleotide to be
replicated.
• DNA turns are added to newly synthesized
circular molecules to introduce supercoiling.
• DNA topoisomerases are able to separate the two
strands of a DNA molecule.
• They can do so without actually rotating the double
helix.
• They achieve this feat by causing transient single- or
double-stranded breakages in the DNA backbone.
• DNA topoisomerases therefore have both nuclease and
ligase activities.
• To carry out blunt end ligation with a topoisomerase, a
special type of cloning vector is needed.
• This is a plasmid that has been linearized by the
nuclease activity of the DNA topoisomerase
enzyme from vaccinia virus.
• The vaccinia topoisomerase cuts DNA at the
sequence CCCTT, which is present just once in
the plasmid.
• After cutting the plasmid, topoisomerase
enzymes remain covalently bound to the
resulting blunt ends.
• The reaction can be stopped at this point,
enabling the vector to be stored until it is
needed.
• Cleavage by the topoisomerase results in 5′-OH
and 3′-P termini.
• If the blunt-ended molecules to be cloned have
been produced from a larger molecule by
cutting with a restriction enzyme, then they will
have 5′-P and 3′-OH ends.
• Before mixing these molecules with the
vector, their terminal phosphates must be
removed to give 5′-OH ends that can ligate to
the 3′-P termini of the vector.
• The molecules are therefore treated with
alkaline phosphatase.
• Adding the phosphatased molecules to the
vector reactivates the bound topoisomerases.
• The enzyme proceed to the ligation phase of
their reaction.
• Ligation occurs between the 3′-P ends of the
vectors and the 5′-OH ends of the phosphatased
molecules.
• The blunt-ended molecules therefore become
inserted into the vectors.
• Only one strand is ligated at each junction
point.
• However, this is not a problem because the
discontinuities will be repaired by cellular
enzymes after the recombinant molecules
have been introduced into the host bacteria.