Home
/
Resources
/
What Is DNA Ligase? Definition & Role

What Is DNA Ligase? Definition & Role

A Brief History

DNA ligase is an enzyme that joins DNA strands through the formation of phosphodiester bonds in a process called DNA ligation. The bond in double-stranded DNA is formed by joining the 5′ phosphate and 3′ hydroxyl termini of DNA strands, using ATP as a coenzyme.

The discovery and characterization of DNA ligase enzymes occurred in 1967 by several laboratories, including Lehman, Gellert, Hurwitz, and Richardson labs. This finding was critical for developing and introducing the concept of molecular cloning and several other future biology experiments.

In organisms, DNA ligase has an essential role in DNA replication and repair. It is also a vital component of in vitro molecular biology and biotech lab assays. It is a member of the nucleotidyltransferase superfamily, along with RNA ligases and mRNA capping enzymes—all of which are essential for maintaining the structural integrity of the genome.

The importance of the enzyme in organisms can be understood by the following example.

Normal cellular processes, including replication, repair, and recombination, often leave phosphate breaks in the genome. This can cause loss of information and introduce deleterious chromosomal mutations. However, DNA ligase prevents these consequences through its enzymatic joining of nucleic acid strands.

In this article, we will cover even more functions of DNA ligase, and the enzymatic ligase reaction mechanisms in organisms and in labs.

Need new or refurbished lab equipment? Excedr leases.

See our equipment list and browse a sample selection of what we can source. Or, if you’re ready, request an estimate.

Types of DNA Ligase

The DNA ligase enzyme has many forms in different organisms, which include:

  • E. coli DNA ligase: Utilizes nicotinamide adenine dinucleotide (NAD) to form a phosphodiester bond between DNA strands. However, it doesn’t join strands with blunt ends. Furthermore, the performance of E. coli DNA ligase can be enhanced under in vitro conditions by adding DNA polymerase in the right amount to the reaction mixture.
  • T4 DNA ligase: Isolated from bacteriophages and is one of the most commonly used DNA ligases for research purposes. It can join oligonucleotides, blunt and cohesive ends of DNA, RNA and RNA/DNA hybrids.

Unlike E. coli, T4 DNA ligase uses adenosine triphosphate (ATP) as a cofactor rather than NAD.

  • Mammalian DNA ligase
  • DNA ligase I: After ribonuclease H removes the RNA primer from the Okazaki fragments, DNA ligase I joins the nascent DNA.
  • DNA ligase II: Produced after DNA ligase III is degraded by proteases (an enzyme that breaks down long polypeptides into short amino acid fragments).
  • DNA ligase III: The only ligase that is present in the mitochondria. It facilitates DNA repair by the process of nucleotide excision repair in association with XRCC1 protein.
  • DNA ligase IV: Associates with XRCC4 protein and facilitates the repair of double-stranded DNA breaks through the non-homologous end-joining (NHEJ) pathway.
  • Thermostable DNA ligase: Found in a thermophilic bacterium and can withstand a higher temperature of 95 Celcius, unlike all other DNA ligases.

How Does DNA Ligase Work?

The enzymatic ligation reaction of DNA ligase follows three unique steps to join two strands of nucleic acid or polynucleotide chains.

After the rearrangement of the activity site’s breaks (or nicks) of DNA fragments (Okazaki fragments), the ligase reaction follows as:

  • 1st step: Release of the pyrophosphate group while adding AMP (adenylation) of a lysine residue (active site of the enzyme) in the DNA ligase enzyme.
  • 2nd step: Formation of the pyrophosphate bond following the transfer of AMP by the DNA ligase enzyme to the 5′ phosphate end of the donor.
  • 3rd step: Formation of phosphodiester bonds when the 5′ phosphate end of the donor chain combines with the 3′ hydroxyl end of the receptor chain.
An illustration of the three-step ligation reaction.
Figure: An illustration of the three-step ligation reaction. Picture Credit: GoldBio.


DNA Ligase Roles & Functions

DNA ligase has several molecular and biochemical functions inside living organisms. Furthermore, they play a huge role in lab experiments by helping biologists understand the dynamics of living bodies.

Uses In organisms

  • DNA replication: DNA ligase joins the strands of DNA formed during the synthesis of new DNA. It forms a phosphodiester bond between the Okazaki fragments of DNA generated on the lagging strand during replication.
  • DNA repairing: DNA ligase has a role in repairing both single-strand nicks and double-strand nicks in duplex DNA. It uses the complementary strand in the double helix as a template and forms a phosphodiester bond to repair single-strand breaks.

Uses In labs

Commercially available DNA ligases have several uses in lab workflows. Of all the available DNA ligases, T4 DNA ligase is most commonly used in lab assays with other reagents, including T4 polynucleotide kinase, tris-HCL, T4 DNA Ligase Reaction Buffer, ATP, glycerol, DTT, and nuclease-free water.

Below are a few common laboratory applications of DNA ligase:

  • cDNA synthesis
  • Circularization of linear DNA
  • Ligation of blunt (DNA strand with no tails or overhangs) and cohesive ends (single-strand DNA with overhangs)
  • Nick repair in single-stranded RNA, DNA, and RNA/DNA hybrids
  • Amplified fragment length polymorphism (AFLP)
  • Cloning of DNA fragments generated by using restriction enzymes
  • Site-direct mutagenesis
  • RNA detection mediated by ligase
  • To build a recombinant plasmid (DNA ligase is used with a restriction endonuclease to insert a foreign gene into a plasmid)

Diseases Caused Due to DNA Ligase Malfunction

DNA ligases are involved in a variety of DNA metabolic pathways. Therefore, their genetic inactivation results in a range of phenotypes or physical appearances, such as hypersensitivity to DNA damaging agents and lethality.

Below are some common diseases caused by dysfunction or lack of DNA ligase:

  • Xeroderma pigmentosum: An inherited condition that makes the infected person sensitive to UV rays. It primarily affects the skin and eyes of the sufferer.
  • Bloom syndrome: In this condition, the affected person is sensitive to the sun. In the exposure to sunlight, the individual develops rashes and red, patched skin on the cheeks and nose.
  • Ataxia-telangiectasia: Occurs due to a mutation in the ATM gene, which is involved in controlling cell division and DNA repair in coordination with the DNA ligase enzyme.

The affected individual faces difficulties in keeping balance, walking, and hand-coordination. Furthermore, they might also have disturbed nerve function and involuntary movements.

Wrap-Up & Takeaways

DNA ligase is an enzyme that facilitates the formation of a phosphodiester bond and joins two DNA strands. Other than DNA synthesis, the enzyme has a critical role in DNA repair in all organisms.

It is also an important substrate for in vitro experiments in molecular biology labs, including molecular cloning, performing PCR reactions, and performing ligation reactions in joining blunt and cohesive ends of DNA.

Reliable and validated data in lab experiments can only be produced when a lab uses high-throughput lab instruments in conjunction with high-quality reagents. However, it is not easy to acquire the right equipment when a lab is tight on funds.

In such situations, Excedr’s leasing program is the most efficient solution. Our program helps labs and organizations of all sizes outfit their labs with desired equipment at an affordable cost. Labs can choose from our extensive range of available instruments, including clinical, biotech, and analytical equipment.