DNA Ligase, T4

T4 DNA ligase is an enzyme encoded by bacteriophage T4. It catalyzes a joining reaction between DNA molecules involving the 3' - hydroxy and the 5' - phosphate termini. It also catalyzes the covalent joining of two segments to one uninterrupted strand in a DNA duplex, provided that no nucleotides are missing at the junction (repair reaction). For its catalytic activity the enzyme requires the presence of ATP and Mg++. DNAs that lack the required phosphate residues can be rendered capable of ligation by phosphorylation with T4 polynucleotide kinase. In addition the enzyme catalyzes an exchange reaction of phosphate between pyrophosphate and ATP. The following is an illustration of the ligation and the repair catlayzed reactions of T4 DNA ligases.

1- Ligation of DNA with complementary cohesive termini

formula

2- Repair reaction

repair reaction

Characteristics of the Enzyme from E. coli lysogenic NM989

Bacteriophage T4 DNA ligase is a single polypeptide with M.W. of 68,000 daltons. Maximal activity is obtained at pH 7.5 - 8.0. At pH 6.9 and pH 8.3 the enzyme exhibits 40% and 65% of its full activity respectively. Mg++ presence is required and the optimal concentration is 10 mM. Sulfhydryl reagents (DTT, 2-mercaptoethanol) are also required. NaCl in concentrations exceeding 200 mM is inhibitory. For intermolecular ligation, especially when the subtrate DNAs consist of large DNA molecules PEG (concentrations of 1 % - 10%) appears to stimulate the enzymatic activity.

Application

T4 DNA ligase is primarily used in joining DNA molecules with compatible cohesive termini, or blunt ended double stranded DNA to one another or to synthetic linkers. Although the reaction involving blunt ended DNA is much slower, the rate of ligation can be accelerated by the additon of 150-200 mM NaCl and low concentration of PEG. Since the 5' - phosphate is essential for the ligase reaction, DNAs that lack the 5' phosphate termini have to be phosphorylated prior to ligation. Phosphorylation is achieved by using T4 polynucleotide kinase and ATP. The joining of DNA fragments with protruding 5' termini that are not compatible (for instance, restriction digestion of DNA with Xba I and Hind III) can be accomplished by partial filling of the recessed 3' termini in controlled reactions using the Klenow fragment of E. coli DNA polymerase I.

Assay

The assay of T4 DNA ligase is based on the capacity of the enzyme to catalyze an exchange reaction between pyrophosphate and ATP. The exchange involves a phosphate molecule from the pyrophosphate and the g phosphate of ATP. One unit ( Weiss unit) of enzyme catalyzes the exchange of 1 nanomole of 32P from pyrophosphate into a Norit absorbable compound in 20 minutes at 37 deg.C.

Reagents

 

Procedure

To clean dry glass test tubes add reagents as follows:

Tris*HCl buffer 0.1 ml
ATP 0.1 ml
Pyrophosphate 0.1 ml
Enzyme sample 0.01 - 0.1 units

Also include a tube without enzymes to be used as a blank. Incubate for 20 minutes at 37deg. C. Stop reaction by adding 2 ml ice cold 2% Norit. Mix suspension on a vortex mixer (Caution, hold tube by the middle when mixing). Keep on ice for 5 minutes. Collect Norit by filtration through glass fiber filter (Whatman GF/C 2.4 cm) with vacuum. Wash four times using 3 ml 0.01 M HCl each time. Transfer filter to scintillation vials. Add 2 ml Cellosolve and mix. Then add 10 ml scintillation liquid and mix. Leave overnight for the Norit to settle and count in scintillation counter set for 32P next day.

Calculation

calculation

 

References

Murray, N., Bruce, S., and Murray, K.: Molecular Cloning of DNA Ligase Gene from Bacteriophage T4. Journ. Molec. Biol., 132, 493 (1979).

Weiss, B., Jacquemin-Sablon, A., Live, T., Fareed, G., and Richardson, C.: Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Further Purification and Properties of Polynucletoide Ligase from Escherichia coli Infected with Bacteriophage T4. J. Biol. Chem., 243, 4543 (1968).

Wilson, G., and Murray, N.: Journ. Molec. Biol, 132, 471 (1979).

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