Structural Biochemistry/T4 DNA Ligase

From Wikibooks, open books for an open world
< Structural Biochemistry
Jump to: navigation, search
DNA ligase shown linking two strands of DNA.

DNA ligase is a special type of ligase that can link two DNA strands that have a break in both complementary strands of DNA together by creating phosphodiester bond. DNA ligase has applications in both DNA repair and DNA In addition. It is used a lot in molecular biology laboratories for genetic recombination experiments. DNA ligases are a useful tool in generating recombinant DNA sequences. For example, DNA fragments are cut with restriction enzymes and then recombined with DNA ligase. DNA ligases are commonly found in E. Coli.


T4 DNA ligase is an enzyme that is encoded by the bacteriophage known as T4. In a reaction where the DNA molecues are being joined together at the 3'-hydroxy and 5'-phosophate termini, the ligase is used as a catalyst. Given that there are no missing nucelotides in the repare reaction, the ligase can also catalyze the covalent joining of two segments to one uniterrupted strand in a DNA duplex. In order to accomplish this catalytic activity, ATP and Mg2+ is required. DNA that lacks the required phosphate residues can still be made to ligate through phosphorylation with T4 polynucleotide kinase. Additionally, an exchange reaction of phosphate between pyrophosphate and ATP can also be catalyzed through use of the ligase.


Characteristics of T4 DNA Ligase from E. coli lysogenic NM989[edit]

The T4 DNA ligase is a single polypeptide with a molecular weight of 68,000 daltons. In order to obtain the maximum amount of activity from the ligase, a pH of 7.5-8.0 is desired. At pH levels of 6.9 and 8.3, the enzyme exhibits 40% and 65% of its full capabilities, respectively. As previously mentioned, Mg2+ is necessary for T4 DNA ligase to be effective, and the optimal concentration of Mg2+ is 10mM. Sulfhydryl reagents (DTT, 2-mercaptoethanol) is also necessary in order to utilize the enzyme. If there is NaCl present, concentrations over 200 mM will stop all enzymatic reactions from occurring. for intermolecular ligation, especially when the substrate DNA consists of large DNA molecules PEG (concentrations of 1-10%) appears to stimulate the enzymatic activity.

Application[edit]

T4 DNA ligase is mostly used in the joining of DNA molecules with compatible cohesive termini, or blunt-ended, double-stranded DNA to one another, or to synthetic linkers. The reaction that involves blund-ended DNA is slower than the other reactions, but the rate of ligation can be accelerated by adding 150 to 200 mM of NaCl along with a low concentration of PEG. If the 5' phosophate is absent in the DNA, then phosphorlation is necessary before ligation can be performed. Phosphorlyation is achieved through utilization T4 polynucleotide kinase with ATP. If the DNA fragments being joined together have protruding 5' termini that are not compatible with one another, it is still able to join the two fragments together by partial filling of the recessed 3' termini in controlled reactions using the Klenow fragment of E. Coli DNA polymerase I.