DNA ligase

Lore & Background

It was first purified and characterized by Weiss and Richardson using a six-step chromatographic-fractionation process. The E. coli DNA ligase is encoded by the lig gene and uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. In contrast, the DNA ligase from bacteriophage T4 is the most commonly used in laboratory research and has an absolute requirement for ATP as a cofactor.

Reader's Guide

DNA ligase is indispensable in molecular biology for generating recombinant DNA sequences, used with restriction enzymes to insert DNA fragments into plasmids. The T4 DNA ligase is most active at 37 °C, but ligation reactions are often performed at 16 °C to balance enzyme activity with stable annealing of sticky ends. For blunt-ended DNA, ligation efficiency is lower and requires higher enzyme concentrations, typically carried out at 14-25 °C overnight. Thermostable DNA ligase, derived from a thermophilic bacterium, is stable at high temperatures and permits extremely high hybridization stringency. DNA ligase also has novel applications in DNA origami for assembling nanoscale structures.

Did You Know?

• DNA ligase IV catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway and is required for V(D)J recombination.
• T4 DNA ligase can ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase.
• Mammals have four specific types of DNA ligase: ligase 1, ligase 3, ligase 4, and ligase 2 (a purification artifact).
• Thermostable DNA ligase has a half-life of 48 hours at 65 °C and greater than 1 hour at 95 °C.