It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. The T4 ligase is the most-commonly used in laboratory research. The DNA ligase from bacteriophage T4 (a bacteriophage that infects Escherichia coli bacteria). When the concentrations of Pol I DNA polymerases are higher, it has an adverse effect on E. Enhancement only works when the concentrations of the DNA polymerase 1 are much lower than the DNA fragments to be ligated. coli DNA ligase can be enhanced by DNA polymerase at the right concentrations. It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. coli DNA ligase is encoded by the lig gene. Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.

Ī pictorial example of how a ligase works (with sticky ends)

Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.

Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond.

Adenylylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released.

Reorganization of activity site such as nicks in DNA segments or Okazaki fragments etc.

AMP is required for the ligase reaction, which proceeds in four steps: Two ATP molecules are consumed for each phosphodiester bond formed. The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide ('acceptor'), with the 5' phosphate end of another ('donor'). The ligase joins the two fragments of DNA to form a longer strand of DNA by 'pasting' them together. Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.Įnzymatic mechanism The image demonstrates how ligase (yellow oval) catalyzes two DNA fragment strands. In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see Research applications). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.ĭNA ligase is used in both DNA repair and DNA replication (see Mammalian ligases). a break in both complementary strands of DNA).

It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. Artist's conception of DNA ligase repairing chromosomal damageĭNA ligase is a type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

Dna ligase and okazaki fragments