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In molecular biology, DNA ligase is a special type of ligase (EC 6.5.1.1) that can link together two DNA strands that have double-strand break (a break in both complementary strands of DNA). The alternative, a single-strand break, is fixed by a different type of DNA ligase 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 two 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 the nascent DNA of the lagging strand after the DNA polymerase I has removed the RNA primer from the Okazaki 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.
Some forms of DNA ligase present in bacteria (usually larger) may require NAD to act as a co-factor whereas other forms of DNA ligases (usually present in E.Coli, and usually smaller) may require ATP to react. Also, a number of other structures present in the DNA ligase are the AMP and lysine which are both important in the ligation process since they create an intermediate enzyme.
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 the ligation of cohesive-ended fragments 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 with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated.[1] If the ambient temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding.[citation needed] The shorter the DNA fragments, the lower the Tm.
Since blunt-ended DNA fragments have no cohesive ends to anneal, controlling the optimal temperature becomes much less important. The most efficient ligation temperature will be the temperature at which T4 DNA ligase functions optimally (T4 DNA ligase is the only commercially-available DNA ligase to anneal blunt ends).[1] Therefore, the majority of blunt-ended ligations are carried out at 20-25°C.
The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli and other bacteria.
See also
References
External links
- DNA Ligase: PDB molecule of the month
- Davidson College General Information on Ligase
- OpenWetWare DNA Ligation Protocol
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