“DNA ligase joins two DNA fragments by forming a phosphodiester bond between them using a molecule of energy.” 

In a routine day, we came across so many adverse conditions such as radiation, high heat, chemicals and mutagens, those agents mutate our DNA or break it. 

But our body’s natural repair mechanism repair damaged DNA, thus, we remain safe or unmutated. 

Many enzymes involved in doing DNA repair and replication.

For instance, DNA polymerase. 

The DNA polymerase adds nucleotides to any gaps occurred in our genome. DNA topoisomerase unwinds DNA for doing replication. Primase like enzyme inserts RNA primers to the replication place. 

Helicase unwinds the DNA double-strand and makes single-stranded DNA for repairing DNA and replicating it.

All these enzymes involved in the DNA repair and replication pathway but one enzyme is even more necessarily required as like all these. 

DNA ligase, 

DNA ligase joins or seals each and every gap by creating the phosphodiester bonds between the two DNA molecules. 

For instance, once DNA polymerase added nucleotides, the gap between the two strands (of two DNA) is filled by DNA ligase. 

In a simple language, we can the ligase is a natural sealant just like a glue that ties two single-stranded ends or two double strands of DNA. 

Read more on DNA: DNA story: The structure and function of DNA

In the present article, we will explain to you the mechanism of how DNA ligase covalently joins two DNA molecules, different types of DNA ligase and their role in genetic engineering. 

Read our article on genetics: Introduction To Genetics: Definition, History, Applications And Branches 

What is DNA ligase? 

The DNA ligase is a class of the enzyme that helps in repairing DNA damage by forming the 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. 

It is involved in the biological processes of 

• Single strand breaks repair 
• Double strand breaks repair 
• DNA replication
• Sticky and blunt end joining 

Furthermore, 

The T4 like DNA ligase is used in the recombinant DNA technology for inserting DNA into the plasmid. 

Mechanism of DNA ligation: 

The 5’ end of the DNA breaks is called “donor” while the 3’ ends are called “acceptor”.

The catalytic reaction of ligation is started with the recognition of the ligation site as nick. The active centre of the enzyme becomes adenylated by addition of AMP to its lysine and starts the enzymatic reaction. 

Here, 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 bond formed between the lysine of enzyme and the AMP is called the phospho-amide bond. 

Once the enzyme attached to the action site called nick, after adenylation the pyrophosphate from the 5’ end of the DNA released from the triphosphate. See the figure below,

The ligase enzyme transfers the AMP to the 5’ phosphate acceptor end. 

Now the enzyme attaches the acceptor to the donor by creating the phosphodiester bond and releases the AMP from the active site. Two phosphate bonds are involved in the formation of phosphodiester bonds between donor and acceptor. 

During the entire reaction, two different ATP molecules are utilized during two different in ligation steps. 

Interestingly, only T4 DNA ligase utilized ATP as an energy cofactor other prokaryotic ligases such as E.coli or bacterial DNA ligase uses NAD⁺ as an energy source. 

Note: Here DNA ligase is not adding any nucleotide to the DNA strand (that can be done only by polymerase), it only fills the gap.

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. 

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.

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.

Read more on DNA replication: General process of DNA 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.

The function of DNA ligase in recombinant DNA technology:

Eukaryotic DNA ligase alike DNA ligase I, DNA ligase II or DNA ligase IV can not 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, 

 

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 basepairs 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.

Different types of DNA ligase: 

Eukaryotic DNA ligase: 

DNA ligase I: ligates the nascent 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 synthesised 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. 

[epcl_box type=”information”]Note: the eukaryotic DNA ligase utilized ATP as a co-factor instead of NAD+.[/epcl_box] 

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 joint double-stranded DNA 
• Ligate DNA- DNA 
• DNA-RNA 
• RNR-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. 

Though it is a “superman” for the recombinant DNA technology, the T4 DNA ligase can not ligate the single-stranded DNA breaks. 

Instead of NAD⁺, it utilizes ATP as an energy molecule for performing the enzymatic reaction. 

Some of the information related to the T4 DNA ligase is given into the table below, 

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

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

Mechanism of T4 DNA ligase: 

The mechanism of T4 DNA ligase is almost the same as the other ligation reaction. However, instead of NAD⁺ used by most prokaryotic ligase, the T4 DNA ligase required the ATP. 

The reaction starts with the nucleophilic attack. 

The AMP attached with the lysine amino acid present in the active site of the enzyme by releasing the PPi. 

In the second step, the active site binds with the 5’ end of the DNA and release the AMP from the lysine and attached it with the phosphate of the donor. 

In the last step, the 5’ donor and acceptor 3’ ends ligated and release the AMP and PPi. 

The temperature vs the activity graph of the T4 DNA ligase is shown here,

In Vitro ligation using T4 DNA ligase: 

A gene expression experiment starts with cloning the gene of interest into the plasmid. 

For doing it, we have to ligate the GOI into the plasmid. 

The first step of obtaining the DNA insert might be the restriction digestion, if we digested our DNA, the end of the DNA surely be stick having few nucleotide overhangs. 

The T4 DNA ligase can ligate the sticky ends of dsDNA very efficiently, therefore, we are using it in recombinant DNA experiment or cloning methods. 

Our selected vectors also have the sticky ends complementary to our digested DNA. However, the plasmid ends can be self-ligated. 

How to prevent self-ligation of plasmid?

If the two ends (two sticky ends) of the plasmid are complementary to each other, it is possible that both the ends fuse to one another and, 

This will reduce the capacity of the plasmid to ligate with insert or gene of interest into it. 

For avoiding the self-ligation in DNA cloning experiment, the selected plasmid is treated with the phosphatase enzyme. 

The phosphatase enzyme removes one of the phosphates from the backbone of the DNA at 5’ end and prevents self-ligation of a plasmid. 

In addition to this, 

Use two different DNA ligase enzyme for two different ends of DNA which will also prevent the self-ligation of a plasmid. 

For achieving ligation perfects use three-part of insert (GOI) and one part of a plasmid (3:1). Although, experts advice must be taken before performing the experiment. 

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 preparation: 

Tris-HCl: 500mM

MgCl2: 100mM

Dithiothreitol: 100mM 

ATP: 10mM

Adjust pH 7.5 store at room temperature. 

Important note for handling the DNA ligation buffer: 

The DNA ligation buffer for T4 DNA ligase contains 10mM part of ATP which provides two ATP energy molecule in each reaction. 

The activity of ATP degrades upon repeated freeze/thawing, even, every sensitive chemical can deteriorate upon repeated freezing and thawing. 

Avoid repeated freezing and thawing, instead, store the buffer in different working aliquotes. 

Always store buffer at -20°C to protect the ATP. 

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 (see the graph above). 

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. 

This DNA ligase can be stably worked for more than 50 thermal cycle reaction. At higher temperatures, a higher ligation specificity and activity can be achieved. 

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 
• It is also useful in nanochemistry and DNA origami experiments.

Although DNA ligase functionally associated with DNA replication and DNA repair, deficiency of any DNA ligase gene leads to some developmental and immunological deficiency. 

 Lack of any DNA ligase results in improper DNA repair. LIG4 syndrome, Fanconi anemia, Ataxia-telangiectasia and bloom syndrome are commonly caused by the deficiency of ligase. 

Tips and tricks: 

Heat the sample for 65°C for 5 minutes before preparing it for a reaction. Remember! Never heat DNA at or over 95°C, it will denature the DNA as well as the plasmid. 

ATP is a heart of the ligation reaction, thus it is very necessary to protect it, as we said earlier avoid repeated freezing and thawing. 

Instead of that, aliquot the buffer for one-time use. 

During the reaction, properly thaw the reaction buffer, do not heat it. Mix it well before use. 

For any molecular biology experiments, the optimum pH is a success factor. 

The ideal pH for the ligation reaction should be between 7.8 to 8.0. In case your experiment failed, check the pH of the reaction buffer. 

At room temperature, it is possible that few bases of DNA-DNA or vector-vector can bind, this will decrease the efficiency of the ligation reaction, heat sample before ligation. 

The success rate of the ligation reaction is very low, you need to develop extraordinary expertise to achieve ligation all the time. 

For that just like playing with the PCR reaction buffer or DNA extraction buffer, you need to add different ligation enhancer chemicals to boost the reaction. 

One of them is the PEG (polyethylene glycol) or you can change the concentration of MgCl₂ to achieve success. 

As we mentioned earlier, use three-part of the insert and one part of the plasmid for the reaction. 

Using control during the reaction is a wise decision. 

Control reaction is very important to conclude why our reaction failed. Prepare a control reaction without using an insert DNA, use only plasmid DNA. 

This will clarify whether the plasmid has a self-ligation capacity of not. As per your expertise, you can use different controls during the reaction to check different parameters. 

Make sure that all of the chemicals and plasticware are used in the experiment must be nuclease-free or DNase free. 

Further, it must not be contaminated with other DNA. 

Pro tips: a blunt end DNA can also be joined using the higher concentration of T4 DNA ligase, the ration for blunt end joint for insert: plasmid must be 10:1. 

Quick FAQs: 

What is DNA ligase? 

DNA ligase is an enzyme required for joining the two DNA ends by creating phosphodiester bonds between them. 

Which DNA ligase is used frequently in the recombinant DNA technology? 

T4 DNA ligase. 

Maximum activity of T4 DNA ligase achieved at which temperature? 

37°C temperature, the activity of the ligase decrease over this temperature. 

Which DNA ligase is active even at a higher temperature?

Ampligase DNA ligase, isolated from the thermophilic bacteria and can actively work at 95°C. 

What is the function of DNA ligase? 

DNA ligase joins or fills the gap between two different DNA molecules. 

Which energy source is required for T4 DNA ligase? 

Two ATP molecules required as a cofactor for T4 DNA ligase. 

Which amino acid is present at the active center of the DNA ligase? 

Lysine. 

Interesting article: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray]

Conclusion:

Conclusive, we can say that DNA ligase is one of the important tools in molecular genetics, especially, in the recombinant DNA creation. The recombinant DNA can be used for creating new species of plant against stresses and to improve the quality of the plant. 

Furthermore, recombinant DNA is also used for the generation of therapeutic drugs and proteins.