2. CONTENT
• Introduction
• Mechanism of DNA ligase
• Unit of enzyme activity
• Types of DNA Ligase:
1)Bacteriophage T4 DNA ligase
2)E.coli DNA Ligase
3)Taq DNA Ligase
4)T4 DNA Ligase
• Applications
• Reference
3. Introduction
• 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
• The DNA ligase used in molecular cloning differ in their
abilities to ligate noncanonical substrate,such as blunt
ended duplexeDNA:RNA hybrid or ssDNAs.
5. Mechanism of DNA Ligase
• The mechanism of DNA ligase is to form two covalent
phosphodiester bonds between 3' hydroxyl ends of one
nucleotide, ("acceptor") with the 5' phosphate end of
another ("donor").
ATP is required for the ligase reaction, which proceeds in three
steps:
(1) adenylation (addition of AMP) of a residue in the active
center of the enzyme, pyrophosphate is released.
(2) transfer of the AMP to the 5' phosphate so-called
donor, formation of a pyrophosphate bond;
(3) formation of a phosphodiester bond between the 5'
phosphate of the donor and the 3' hydroxyl of the acceptor.
6.
7. • Depending up on the source,the enzyme requires
either ATP or NAD+ as cofactors
8. Unit of Enzyme Activity
• For all ligase, one Weiss unit is the amount of
Enzyme required to convert 1 n mol of radiolabeled
phosphate from pyrophosphate into absorbable
material in 20 min at 370 c under standard assay
condition.
one Weiss unit equals about 67 cohesive end ligation
unit.
9. 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.
10.
11. 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
12.
13. E.Coli DNA ligase
• It is derived from E.coli 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.
14. Application:
• 1) ligation of cohesive ends:E.coli DNA ligase is used
to catalyze sticky end ligation.
• 2)cloning of full length cDNA:
E.coli DNA ligase has been employed in a procedure
for high efficiency cloning of full length cDNA
15. Taq DNA ligase [NAD+ ]
The gene encoding thermostable ligases have
been identified from several thermophilic
bacteria.
Several of this ligase have been cloned and
expressed to high levels in E.coli
• It is uses in the detection of mutation as
thermostable DNA ligase retain their activities after
exposure to higher temp for multiple rounds
• it is uses in DNA amplificaton reaction to detect
mutation in mammalian DNA.
16. T4 RNA Ligase
• T4 RNA ligase is the only phage RNA ligase that has
been extensively characterized and used in genetic
engineering.
• This enzyme catalyzed the phosphodiester bond
formation of RNA molecule with hydrolysis of ATP to
PPI
• It is monomeric enzyme with 373 deduced amino
acid residues is a product of the T4 gene 63
17. Application
1) 5’-Tailing of DNA with RNA
2) Radiolabeling of 3’ end of RNA and DNA
3) production of elongated molecules
4) modification of internal Nucleotide
5) stimulation of T4 DNA ligase activity
18. .
Mammalian ligases
.
DNA ligase I: ligates the nascent DNA of the lagging strand after the
DNA polymerase I has removed the RNA primer from the Okazaki
fragments.
DNA ligase II: alternatively spliced form of DNA ligase III found in
non-dividing cells.
DNA ligase III: complexes with DNA repair protein XRCC1 to aid in
sealing base excision mutations and recombinant fragments.
DNA ligase IV:. It catalyzes the final step in the non-homologous end
joining DNA double-strand break repair pathway. It is also required
for V(D)J recombination, the process which generates diversity in
immunoglobulin and T-cell receptor loci during immune system
development.
