A ligase is an enzyme that can form a new chemical bond to catalyse the fusing of two large molecules through an accompanying hydrolysis of a small pendant chemical group. Ligases use ATP to form the bonds, and is used in recombinant DNA cloning, joining two complementary fragments of nucleic acid.
DNA ligase enzymes carry the EC number 220.127.116.11 (CAS number 9015-85-4) and are used for the joining of two DNA strands by repairing single-strand breaks to catalyse phosphodiester bonds. The relationship occurs between the 5' phosphate end of the donor nucleotide and the 3’ hydroxyl ends of the acceptor nucleotide.
The four-step process, which requires AMP, involves: the reorganization of activity in DNA segments or Okazaki fragments, adenyltation of a lysine residue to the active centre of the enzyme to release the pyrophosphate, transfer of AMP to the donor nucleotide’s 5’ phosphate end to form a pyrophosphate end, formation of the phosphodiester bond between the donor nucleotide and the 3' hydroxyl of the acceptor.
Encoded by the lig gene, The E.coli DNA ligase gains energy by cleaving the nicotinamide adenine dinucleotide (NAD) to generate a phosphodiester bond, but cannot join RNA to DNA. DNA polymerase concentrates can be used to heighten the activity of the E.coli DNA ligase but the concentrates must be smaller than the fragments to be ligated.
The Escherichia virus T4 is primarily used in lab research, and can be ligated to either the cohesive or blunt ends of oligonucleotides in addition to both RNA and RNA-DNA hybrids. However, it doesn't join single-stranded nucleic acid or use NAD. T4 relies on an ATP cofactor and has an optimal temperature of 16 °C.
The thermostable enzyme is derived from the thermophilic bacterium and activates at far greater temperatures than other DNA ligases, underlined by half-life rates of over 1 hour at 95 °C and 48 hours at 65 °C. It permits high hybridization stringency and ligation specificity, showing activeness for 500 thermal cycles.