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Concept
DNA ligase
Summary
DNA ligase is a type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. 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. a break in both complementary strands of DNA). 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.
DNA ligase is used in both DNA repair and DNA replication (see Mammalian ligases). In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see Research applications). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA.
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"). Two ATP molecules are consumed for each phosphodiester bond formed. AMP is required for the ligase reaction, which proceeds in four steps:
Reorganization of activity site such as nicks in DNA segments or Okazaki fragments etc.
Adenylylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released;
Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond;
Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor.
Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.
The E. coli DNA ligase is encoded by the lig gene. DNA ligase in E. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently.
Official source
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