DNA ligase

DNA ligase is an essential enzyme responsible for joining DNA strands by catalysing the formation of phosphodiester bonds. It plays a critical part in maintaining the integrity of the genome, participating in DNA replication, DNA repair, and a wide range of laboratory techniques used to manipulate genetic material. By sealing breaks in DNA, ligases help preserve genetic continuity and support vital processes in both prokaryotic and eukaryotic cells.

Biological Functions of DNA Ligase

In living organisms, DNA ligase repairs single-strand breaks using the intact complementary strand of the double helix as a reference. After other enzymes prepare the site of damage, DNA ligase seals the remaining gap by restoring the phosphodiester linkage. Certain forms of the enzyme are also involved in repairing double-strand breaks, a more serious form of damage in which both strands of the duplex are disrupted. In these cases, specific ligases such as mammalian ligase IV function as part of the non-homologous end joining pathway.
During DNA replication, ligase plays a crucial role in sealing Okazaki fragments on the lagging strand. After RNA primers are removed and replaced with DNA, the newly synthesised DNA fragments require ligation to produce a continuous strand.

Enzymatic Mechanism

The catalytic activity of DNA ligase involves the formation of two new phosphodiester bonds. The reaction consumes ATP in eukaryotes and many viruses, while most prokaryotic ligases use nicotinamide adenine dinucleotide (NAD) as the energy source. The reaction proceeds in a stepwise manner:

• Recognition of irregularities such as nicks or breaks in the DNA backbone
• Adenylylation of an active-site lysine residue, releasing pyrophosphate
• Transfer of the AMP group to the 5′ phosphate of the donor strand
• Creation of the phosphodiester bond linking the 5′ phosphate to the 3′ hydroxyl of the acceptor strand

This mechanism ensures precise repair of DNA and supports accurate duplication of genetic material.

Types of DNA Ligase

Prokaryotic LigasesThe Escherichia coli ligase, encoded by the lig gene, uses NAD as its energy source. It is effective at joining cohesive ends but inefficient at blunt-ended ligation without molecular crowding agents such as polyethylene glycol. It also works poorly with RNA–DNA hybrids. Its activity can be modulated by DNA polymerase I, although excessive polymerase concentrations inhibit the ligase.
T4 DNA LigaseDerived from bacteriophage T4, this enzyme is widely used in molecular biology. It requires ATP rather than NAD and can ligate cohesive and blunt ends with high efficiency. It is also capable of joining DNA–RNA hybrid molecules. Engineering efforts have produced enhanced variants, including ligases fused with DNA-binding proteins, which show improved activity in blunt-end ligation.
T4 ligase is most active at 37 °C, but ligation reactions are often conducted at 16 °C to balance enzyme activity with the stability of annealed cohesive ends.
Mammalian LigasesMammals possess four primary DNA ligases:

• Ligase I, which completes lagging-strand synthesis by sealing Okazaki fragments
• Ligase III, typically associated with XRCC1 for single-strand break repair and found also in mitochondria
• Ligase IV, which partners with XRCC4 to perform key steps in double-strand break repair and is required for V(D)J recombination in immune system development
• Ligase II, now known to be a proteolytic fragment of ligase III and not a unique ligase type

Eukaryotic ligases use ATP as their energy source, reflecting a fundamental biochemical difference from many prokaryotic enzymes.
Thermostable LigasesLigases from thermophilic organisms retain their activity at elevated temperatures. Some remain active for hundreds of thermal cycles, making them valuable in applications requiring precise hybridisation and high sequence specificity.

Measurement of Ligase Activity

Enzyme activity may be quantified using several units:

• Weiss units, the most commonly used, measure AMP–pyrophosphate exchange
• Modrich–Lehman units, which assess conversion of labelled polynucleotides into nuclease-resistant forms
• Various commercial units, which are based on ligation efficiency but often lack standardisation

Such measurements ensure appropriate enzyme usage in experimental conditions.

Applications in Molecular Biology

DNA ligases are indispensable in recombinant DNA technology. They are routinely used to join DNA fragments generated by restriction enzymes, enabling the insertion of genetic sequences into plasmids and other vectors. Temperature conditions must be carefully controlled, as the stability of sticky ends and the efficiency of blunt-end joining depend on thermal compatibility with base pairing and fragment alignment.
For cohesive ends, the selected temperature must support stable annealing without disrupting hydrogen bonds. Blunt-end ligation relies on sufficient molecular collisions, so reactions are often incubated at lower temperatures for extended periods.
Beyond cloning, DNA ligase has emerging roles in nanotechnology, particularly in DNA origami, where it helps assemble complex nanoscale structures by sealing designed DNA frameworks.

Historical Development

The first DNA ligase was isolated in 1967 in studies involving E. coli. Early purification strategies involved multi-step chromatographic separation, revealing that ATP and magnesium ions were crucial for enzymatic activity. These foundational discoveries paved the way for extensive use of DNA ligase in genetic engineering and biotechnology.