In molecular biology, the specific form of the enzyme to promote binding of the DNA strands together, DNA ligase, a ligase to catalyze the formation of phosphodiester bonds. It plays a role in repairing single strand breaks in the double DNA in an organism, but some embodiments, two of the DNA (i.e. double strand breaks (e.g., such as DNA ligase IV) specific You can modify the stop) of the complementary strand of one. Single-strand breaks is carried by DNA ligase and the complementary strand of the double helix as a template and link establishment host total recovery of DNA ligase end of DNA. DNA ligase, has applications in both (see mammalian ligase) DNA replication and DNA repair. There were also widely used in molecular biology laboratory in genetic recombination experiments (see the application of molecular biology) DNA ligase. DNA ligase is purified, used for cloning of the gene that bind the DNA molecules together to form a recombinant DNA.
In mammals, there are certain types of four ligase. DNA ligase I is: ligation the DNA of the lagging nascent chains after being pulled from the primer RNA Okazaki fragments ribonuclease H. It helps to seal the DNA complex with the DNA repair protein XRCC1, in the course of a recombinant fragment and nucleotide excision repair: DNA ligase III. Complex and XRCC4: DNA ligase IV. Join the DNA double strand break repair pathway, which catalyzes the last step of the non-homologous end. Further, it is necessary to produce the various processes of the T cell receptor loci and immunoglobulin during the development V (D) J recombination immune system.
To create a phosphodiester bond, DNA ligase in E. coli, using the energy cut nicotinamide adenine dinucleotide (NAD) origin. Instead of adenosine triphosphate (ATP), eukaryotic microbes and other DNA ligase is using the NAD. Further, because they create intermediate enzymes, various structures of the other present in the DNA ligase is a lysine and AMP which is important for the consolidation.
DNA ligase, has become an essential tool in the study of modern molecular biology for the production of recombinant DNA sequences. For example, often, DNA ligase, used with restriction enzymes to insert a DNA fragment into the gene of the plasmid.
Management optimum temperature is an important aspect for implementing the recombination experiments effective, including binding fragments single type. Most experiments that (separation from T4 bacteriophage) T4 DNA ligase is most active at 25 ℃, to use however, for optimum efficiency, optimum of the enzyme type of single ligation fragment in (“sticky ends”) (annealing temperature) and melting temperature Tm of the sticky ends that you need to balance between the right temperature degree is joined. Tm above the ambient temperature of the homologous binding sticky end because the distorted hydrogen bonds high temperature is not stable. The ligation reaction at the end of curing, sticky is the most effective if already well – the destruction of the firing of all, therefore, would result in lower ligation efficiency. Short overhangs, low CT. In general, four-base overhang is Tm ℃ 12-16
Since blunt end DNA fragments anneal to sticky ends, melting temperature is not a factor in the normal temperature of the ligation reaction. However, the possibility of higher temperature, edge joining lower would be brought in order to allow the connection (moving more in solution at higher temperatures molecule). Instead of ligase activity, limiting factor blunt end ligation, the number of paths across the DNA fragments generated. Therefore, it temperatures most efficient for the ligation of blunt-ended DNA becomes a temperature that may cause multiple paths occurs. Most of the blunt-end ligation is carried out overnight at 14 ~ 20 ℃. Lack of end annealed stable means that the ligation efficiency and need to use higher concentrations ligase is reduced. (T4 DNA ligase, DNA ligase is a commercially available only annealing the blunt end).
DNA ligase, E. coli, is a polypeptide of 75,000 molecular weight. It is slightly smaller (68 000-63 000) in T4-induced enzyme comparable. DPN (E. coli), or both enzymes, phospho between the 3′-hydroxyl group and the 5 ‘phosphoryl adjacent to the double-stranded DNA was nicked coupled to the cleavage of the pyrophosphate bond of ATP (T4) I catalyzes the synthesis of bonds. Phosphodiester bond synthesis, requires the participation of two covalent intermediate and is catalyzed by two enzymes steps series of these individual. The steady-state kinetic analysis of the reaction catalyst E. coli ligase, and supports this mechanism to be important intermediates kinetically for DNA-adenylate and enzyme adenylate, synthetic shortcuts phosphodiester bond It further shows.
Strains of mutation of a structural gene E. coli, the DNA ligase is non-viable at 42 ℃ result of the synthesis of abnormal heat-labile enzyme. It can not grow at 30 ℃, but for their ability to repair DNA damage caused by alkylating agents and ultraviolet light still, there is a defect in the temperature variant. At 42 ℃, “fragment Okazaki” in the form of 10S DNA that has been replicated new is short, keep the ligation process that the cause of the failure of the mutants to survive under these conditions can disrupt the synthesis is essential EF chromosome means that it is that it can not be. DNA ligase is an enzyme essential for normal replication and DNA repair in Escherichia coli. DNA ligase is purified proves reagents useful in the construction of the in vitro recombinant DNA molecule.