DNA-Ligases:
The final step in construction of a
recombinant DNA molecule is the joining together of the vector molecule and the
DNA to be cloned. This process is referred to as ‘ligation’, and the enzyme
that catalyzes the reaction is called DNA-ligase. Thus, DNA ligases is a group
of enzymes which mediate annealing, sealing and joining of DNA fragments.
These enzymes are widely used in genetic
engineering for the production of hybrid or recombinant DNA. Since ligases join
DNA fragments or seal the nick chain, they are called molecular sutures.
The role of DNA ligase is to seal nicks in
the backbone of double-stranded DNA after lagging strand formation to join the
okazaki fragments.
This joining process is essential for the
normal synthesis of DNA and for repairing damaged DNA. It has been exploited by
genetic engineers to join DNA chains to form recombinant DNA molecules. Usually
single stranded break are repaired using the complimentary strand as the
template but sometimes double stranded breaks can also be repaired with the
help of DNA ligase.
DNA
ligase catalyses the formation of phosphodiester bond between two
deoxynucleotide residues of two DNA strands.
DNA ligase enzyme requires a free hydroxyl
group at the 3´ -end of one DNA chain and a phosphate group at the 5´-end of
the other and requires energy in the process.
E.coli and
other bacterial DNA ligase utilizes NAD+ as energy donor, whereas in T4
bacteriophage, T4 DNA ligase uses ATP as cofactor. Except this, the catalysis
mechanism is somewhat similar for both the ligases. The role of cofactor is
splitting and forming an enzyme-AMP complex which further aids in formation of
phosphodiester bonds between hydroxyl and phosphate groups by exposing them.
The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs ATP as a cofactor.
Terminal deoxynucleotidyl transferase enzyme:
TdT enzyme is an template
independent DNA polymerase.
It adds single stranded sequences to
3′-terminus of the DNA molecule i.e., it catalyses the repetitive addition of
mononucleotide units from deoxynucleoside triphosphate(dNTP) to the terminal
3’- hydroxyl group of a DNA molecule. One or more deoxynbonucleotides (dATP, dGTP,
dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments.
If the restriction enzyme cuts
DNA forming blunt ends, then efficiency of ligation is very low. So the enzyme
terminal transferase converts bunt end into sticky end.
Cobalt is necessary co factor for this
enzyme.
Applications:
Terminal transferase is used
to add homopolymer tails of DNA fragments by technique called homopolymer
tailing. For example, one preparation of DNA could be treated with the enzyme
terminal transferase in the presence of dATP, resulting in the addition of a
poly(dA) chain to the 3 end of each strand.
The other preparation would
then be given 3’ tails of poly (T), using the same enzyme with TTP. On mixing,
there would be base pairing between complementary sticky ends, which could then
be ligated. An interesting feature of
this method is that ligation will not occur between fragments from the same
preparation.
It is used for 3’- end
labelling of DNA fragments.
(b) Alkaline Phosphatase Enzyme:
It functions to remove the phosphate
group from the 5′-end of a nucleic acid molecule ( RNA, DNA or ribo- and
deoxyribonucleotide triphosphates).
This enzyme is a dimeric glycoprotein
with a molecular weight of 14,000. It is made up of two identical or similar
subunits each with a molecular weight of 69,000. It is a zinc containing enzyme
with four atoms of Zn2+ per molecule.
Applications:
Linearized cloning vectors can be prevented from recircularizing by dephosphorylation with alkaline phosphatase.
The free 5’-OH can be phosphorylated
with polynucleotide kinase and ϒ- 32P ATP to produce 32P
end labelled nucleic acids.
Enzyme is used for mapping and finger
printing studies.
(c) Polynucleotide Kinase
Enzyme:
It catalyses the transfer of the
terminal phosphate group of ATP to the 5’- hydroxylated terminal of DNA or RNA.
So, it has an effect reverse to that of Alkaline Phosphatase, i.e. it functions to add phosphate group to the
5′-terminus of a nucleic acid molecule.
This enzyme is frequently used to
end-label nucleic acids with 32P.
This 5’-terminal labelling is used in
mapping of restriction sites, DNA or RNA fingerprinting, hybridizations studies
and sequence analysis of DNA.
T4 polynucleotide kinase is the most
widely used PNK in molecular cloning experiments, which was isolated from T4
bacteriophage infected E.coli.
Methyltransferase or methylase
Methyltransferase
or methylase catalyzes the transfer of methyl group (-CH3) to its substrate.
The process of transfer of methyl group to its substrate is called methylation.
Organisms that make restriction enzymes also
synthesis DNA methyltransferase that protects their own DNA from cleavage. These
enzymes recognize the same DNA sequence as the restriction enzyme they
accompany.
Methylation is the process of addition of
methyl groups to adenine or cytosine bases within the recognition site and
thereby modifying the site and prevent the DNA for restriction activity.
Methyltransferase
uses a reactive methyl group that is bound to sulfur in Sadenosyl methionine
(SAM) which acts as the methyl donor. Methylation normally occurs on cytosine
(C) residue in DNA sequence. DNA methylation regulates gene or silence gene
without changing DNA sequences, as a part of epigenetic regulation.
In
bacterial system, methylation plays a major role in preventing their genome
from degradation by restriction enzymes. It is a part of restriction –
modification system in bacteria.
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