DNA ligase

In molecular biology, DNA ligase is a particular type of ligase (EC 6.5.1.1) that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA). The alternative, a single-strand break, is easily fixed by DNA polymerase using the complementary strand as a template but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA.

DNA ligase has applications in both DNA repair and DNA replication (see Mammalian ligases). In addition, DNA ligase has extensive use in molecular biology laboratories for Genetic recombination experiments (see Applications in molecular biology research).

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Ligase mechanism

The mechanism of DNA ligase is to form covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. ATP is required for the ligase reaction.

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

Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.

Mammalian ligases

In mammals, there are four specific types of ligase.

• DNA ligase I: ligates Okazaki fragments during lagging strand DNA replication and some recombinant fragments.
• DNA ligase II: alternatively spliced form of DNA ligase III found in non-dividing cells.
• DNA ligase III: complexes with DNA repair protein XRCC1 to aid in sealing base excision mutations and recombinant fragments.
• DNA ligase IV: complexes with XRCC4. It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway. It is also required for V(D)J recombination, the process which generates diversity in immunoglobulin and T-cell receptor loci during immune system development.

Applications in molecular biology research

DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.

One vital, and often tricky, aspect to performing successful recombination experiments involving ligase is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4) which is most active at 25°C. However in order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature T_m (also the annealing temperature) of the DNA fragments being ligated.

If the ambient temperature exceeds T_m, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. The shorter the DNA fragments, the lower the T_m. Thus for sticky ends (overlaps) less than ten base pairs long, ligation experiments are performed at very low temperatures (~4-8°C) for a long period of time (often overnight).

The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli or other bacteria.

See also

• DNA end
• Lagging strand
• DNA replication
• Okazaki fragment
• DNA Polymerase