Ligase enzyme

1. DNA ligase
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. The main enzymes involved in genetic engineering.
The enzymes are:
1. Restriction Endonuclease
2. DNA Ligase
3. Alkaline Phosphatase
4. Polynucleotide kinase
5. DNA Polymerase and the Klenow Fragment
6. Reverse Transcriptase
7. DNase
8. Rnase
9. DNA polymerase
Enzymes involved in genetic engineering

3. DNA ligase

4. What is DNA ligase?
• The DNA ligase is a class of the enzyme that helps in the formation of phosphodiester bond between the 5’ end
of one side to the 3’ end of another side using an energy molecule (ATP or NAD+).
• First DNA ligase enzyme was purified and characterized by Weiss and Richardson in 1967.
• 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.
The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs ATP as a cofactor.
• The enzyme from E. coli uses cofactor NAD.

5.  All the eukaryotic enzyme works efficiently at body temperature (37°C). However, some of the DNA ligases like the T4
DNA ligase work efficiently at 16°C temperature, in vitro. Even, the ligation can also be achieved at 4°C during in Vitro
reaction.
Components of DNA ligase enzyme:
The T4 DNA ligase contains three parts made up of
the single polypeptide chain.
• Nucleotide-binding site
• DNA binding cleft in the centre
• An active site containing the lysine residue

6. Mechanism of DNA ligation:
1. The 5’ end of the DNA breaks is called “donor” while the 3’ ends are
called “acceptor”.
2. The catalytic reaction of ligation is started with the recognition of the
ligation site as nick.
3. The reaction starts with the nucleophilic attack.
In the first step, one ATP or NAD+ (in case of bacterial ligase) energy
molecules react with ligase enzyme and helps in forming the enzyme-
AMP complex linked to amino group of the lysine. The AMP attached
with the lysine amino acid present in the active site of the enzyme
by releasing the PPi. Thus, the active centre of the enzyme
becomes adenylated by addition of AMP to its lysine and starts
the enzymatic reaction. The bond formed between the lysine of
enzyme and the AMP is called the phospho-amide bond.
In the second step, the active site of the enzyme with the AMP moiety
activates the phosphate group at the 5´-end of the DNA molecule to be
joined [and releases the AMP from the lysine and attached it with the
phosphate of the donor]. Thus the ligase enzyme transfers the AMP to
the 5’ phosphate end.
The final step is a nucleophilic attack by the 3´-hydroxyl group on this
activated phosphorus atom. Now the enzyme attaches the 5’ to the 3’
by creating the phosphodiester bond and releases the AMP from the
active site.

7. Different types of DNA ligase:
Eukaryotic DNA ligase:
 DNA ligase I: ligates the DNA on the lagging strand, especially, the gaps between the Okazaki fragments.
 DNA ligase II: The DNA ligase II is not considered as a true ligase because it does not have its own gene, the
eukaryotic DNA ligase II synthesized from the gene that encodes the DNA ligase III.
It is majorly involved in the DNA repair pathway.
 DNA ligase III: it is required in the DNA repair, especially, in the nucleotide excision repair. It is the only kind of
ligase that present in mitochondrial DNA too.
 DNA ligase IV: The DNA ligase IV is very special because it joints the double-stranded DNA. It is involved in the
double-strand break repair pathway, particularly, in the non-homologous end-joining.
Furthermore, it is also required for the V(D)J fragment joining.
Note: the eukaryotic DNA ligase utilized ATP as a co-factor instead of NAD+.

8. Prokaryotic DNA ligases:
E. coli DNA ligase:
 The E. coli DNA ligase uses NAD+, nicotinamide adenine dinucleotide as a cofactor or energy source for forming the
phosphodiester bond.
 It can join only DNA- DNA molecule and unable to join DNA-RNA. In addition to this, only sticky ends can be sealed by
E.coli DNA ligase.
 The E. coli DNA ligase is encoded by the “lig” gene of E. coli.
T4 DNA ligase:
 In 1083, Armstrong and coworkers discovered the structure and genetic organization of T4 DNA ligase, however, the first
T4 DNA ligase was cloned by Wilson and Murray in 1979.
 The T4 DNA ligase is extracted from the bacteriophage T4, and it is one of the most commonly used DNA ligases in
recombinant DNA technology.
 It is one of the best enzymes for research labs because of its tremendous advantages over E. coli DNA ligase.
 It can join double-stranded DNA
 Ligate DNA- DNA
 DNA-RNA
 RNA-RNA molecule
 Also, it can capable of ligating sticky ends as well as blunt ends.
 The optimum activity of it can be achieved at a lower temperature.
 Instead of NAD+, it utilizes ATP as an energy molecule for performing the enzymatic reaction.

9. Protein Accession
number
P00970
Gene Gp30 (gene 30) of T4 bacteriophage
pH 7.8-8.0
Molecular weight
77 kDa (Sedimentation, Lehman 1974)
74 ± 3 kDa (SDS Page, Lehman 1974)
55.3 kDa (Theoretical)
Isoelectric point 6.14
Activators ATP, Mg2+, sulfhydryl reagents
Optimum temperature 16°C
Inhibitors A higher concentration of NaCl
Information related to the T4 DNA ligase
The temperature vs the activity graph of the T4 DNA
ligase is shown here,

10. The function of DNA ligase:
The main function of DNA ligase is to ligate two DNA strands either single strand
or double strand. Although, different ligase is used for different function in vivo
and in vitro processes.
1. The function of DNA ligase in Replication:
 Replication is a process in which the four different daughter single-stranded DNA
molecules generated from a single DNA duplex (dsDNA). Different enzymes
work in different steps for completion replication.
 The process of DNA replication starts with the addition of RNA primer by the
primase enzyme. The 3′ end of the primer is used as a starting point for the
addition of nucleotides by the DNA polymerase at the leading strand.
 The replication ends at the lagging strand by the synthesis of Okazaki fragments.
 In the final steps, the primer is removed and the nucleotides are filled in the gaps
between the Okazaki fragments by DNA polymerase but the newly synthesised
strands are still not joined.
 DNA Ligase performs the function of fillings gaps by creating phosphodiester
between the gaps, created after the removal of primer and between the Okazaki
fragments.
 It performs ligation by using the 5′ end of the one strand and 3′ end of another
end and joins it by removing the pyrophosphate from the triphosphate.
 However, the ligase used in the DNA replication can not ligate the double-
stranded DNA or blunt-ended ds DNA.

11. The function of DNA ligase in recombinant DNA technology:
 Eukaryotic DNA ligase alike DNA ligase I, DNA ligase II or DNA ligase IV cannot be used in the cloning experiments,
instead, the phage T4 DNA ligase is used for performing different ligation methods.
 Restriction digestion of DNA generates two types of DNA ends viz sticky ends or blunt ends.
 Different ends are generated for different molecular biology techniques.
Process of blunt-end ligation:
 Blunt ends are generated due to the restriction digestion at the same base pair on both these strands.
 The ends are simple and, direct and noncohesive. Special types of DNA ligase are used to ligate these types of DNA ends.
The process of blunt-end ligation as shown into the figure below,

12. Process of sticky end ligation:
 Restriction digestion which generates few nucleotide overhangs on both the DNA strands is called sticky ends.
 These ends are cohesive and have few base pairs palindromic sequences on both the strands (most cases).
The process of sticky end ligation is shown in the figure below:
The sticky ends are generated for inserting a gene of interest in the plasmid. It works better in comparison with
the blunt end ligation.

13. Preparation of ligation reaction:
Insert DNA: 100ng
Plasmid DNA: ~30ng
T4 DNA ligase: 0.5 to 1μl
Ligation buffer: 10X
D/w total volume of 10μl.
10X ligation buffer Composition:
Tris-HCl: 500mM
MgCl2: 100mM
Dithiothreitol: 100mM
ATP: 10mM
Adjust pH 7.5 store at room temperature.
Incubation:
 The time and temperature for incubation of ligation
reactions are very crucial. All the DNA ligases are generally
active at room temperature, however, the optimum activity
of DNA ligase obtained at 16°C temperature.
 You can incubate the sample at room temperature for 2
hours or at 16°C overnight.
Interestingly,
 One of the commercially available DNA ligase called
“Ampligase” DNA ligase can work efficiently even at a
higher temperature of more than 95°C.
 The “ampligase” is isolated from the thermophilic bacteria
having a half-life of 48 hours at 65°C and 1 hour at 95°C.

14. Application of DNA ligase:
 Ligates the cohesive blunt ends as well as sticky ends.
 Ligate single-stranded as well as double-stranded DNA.
 Used in a ligase chain reaction
 Used during the DNA repair mechanism
 Use during DNA replication
 Used to insert a gene in the plasmid

15. Ligation techniques
1. Ligation dependent Cloning (LC)- using T4 DNA ligase/ E.coli DNA ligase
2. Ligation Independent Cloning (LIC)
• LIC is a cloning method that makes use of annealing of single-stranded complementary overhangs on the
target vector and a PCR-generated insert of at least 12 bases overlap region.
• Single-stranded overhangs can be generated by using T4 DNA polymerase and only one dNTP in the
reaction mix, leading to an equilibrium of 3'->5'-exonuclease and 5'->3'-polymerase activity at the site of the
first occurrence of this nucleotide.
• The incubation is done with T4 DNA pol. for 30 min. and stopped by adding dCTP to the reaction mix. After
annealing of vector and insert, the mixture is used to transform E. coli.
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