Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).
The discovery of DNA ligase dates back to 1967 and is an important event in the field of molecular biology. Ligation in the laboratory is normally performed using T4 DNA ligase. It is broadly used in vitro as molecular biology research tool due to its capability of joining as sticky as blunt DNA ends. However, procedures for ligation without the use of standard DNA ligase are also popular. Human DNA ligases abnormalities have been linked to pathological disorders characterized by immunodeficiency, radiation sensitivity, and developmental problems.
Ligation reaction
The mechanism of the ligation reaction was first elucidated in the laboratory of I. Robert Lehman. Two fragments of DNA may be joined by DNA ligase which catalyzes the formation of a phosphodiester bond between the 3'-hydroxyl group (-OH) at one end of a strand of DNA and the 5'-phosphate group (-PO4) of another. In animals and bacteriophages, ATP is used as the energy source for the ligation, while in bacteria, NAD+ is used.
The DNA ligase first reacts with ATP or NAD+, forming a ligase-AMP intermediate with the AMP linked to the ε-amino group of lysine in the active site of the ligase via a phosphoramide bond. This adenylyl group is then transferred to the phosphate group at the 5' end of a DNA chain, forming a DNA-adenylate complex. Finally, a phosphodiester bond between the two DNA ends is formed via the nucleophilic attack of the 3'-hydroxyl at the end of a DNA strand on the activated 5′-phosphoryl group of another.
A nick in the DNA (i.e. a break in one strand of a double-stranded DNA) can be repaired very efficiently by the ligase. However, a complicating feature of ligation presents itself when ligating two separate DNA ends as the two ends need to come together before the ligation reaction can proceed. In the ligation of DNA with sticky or cohesive ends, the protruding strands of DNA may be annealed together already, therefore it is a relatively efficient process as it is equivalent to repairing two nicks in the DNA. However, in the ligation of blunt-ends, which lack protruding ends for the DNA to anneal together, the process is dependent on random collision for the ends to align together before they can be ligated, and is consequently a much less efficient process. The DNA ligase from E. coli cannot ligate blunt-ended DNA except under conditions of molecular crowding, and it is therefore not normally used for ligation in the laboratory. Instead the DNA ligase from phage T4 is used as it can ligate blunt-ended DNA as well as single-stranded DNA.
Factors affecting ligation
Factors that affect an enzyme-mediated chemical reaction would naturally affect a ligation reaction, such as the concentration of enzyme and the reactants, as well as the temperature of reaction and the length of time of incubation. Ligation is complicated by the fact that the desired ligation products for most ligation reactions should be between two different DNA molecules and the reaction involves both inter- and intra-molecular reactions, and that an additional annealing step is necessary for efficient ligation.
The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. Mg(2+) is a cofactor for catalysis, therefore at high concentration of Mg(2+) the ligation efficiency is high. If the concentration of Mg(2+) is limited, the nick- sealing is the rate- limiting reaction of the process, and adenylylated DNA intermediate stays in the solution. Such adenylylation of the enzyme restrains the rebinding to the adenylylated DNA intermediate comparison of an Achilles' heel of LIG1, and represents a risk if they are not fixed.
