Thermus DNA Ligase

Source: E. coli strain containing an over-producing clone of Thermus DNA Ligase^(1-2).

Applications:

• High temperature ligation
• High fidelity ligation
• Site directed mutagenesis

Thermus DNA Ligase catalyzes the formation of a phosphodiester bond in duplex DNA containing adjacent 5'-phosphoryl and 3'-hydroxyl termini. Thermus DNA Ligase uses nicotinamide adenine dinucleotide (NAD⁺) instead of ATP as a cofactor. Following the ligation reaction, AMP and nicotinamide mononucleotide (NMN) are released.

The ligation mechanism occurs in three steps. First, an adenylyl group (AMP) is transferred from NAD⁺ to the ε-amine group of the active-site lysine residue in the enzyme. This results in the release of NMN. Second, the adenylyl group is transferred from the enzymenucleotide intermediate to the 5'-phosphate of the DNA which activates it for nucleophilic attack. In the third and final step, a phosphodiester bond is formed by nucleophilic attack of the 3'-hydroxyl group of the DNA on the activated 5'-phosphate, which releases AMP⁽³⁾. Thermus DNA Ligase greatly prefers matched duplex DNA over mismatched, particularly within 8 base pairs of the adjacent nicked termini. Thermus DNA Ligase will not ligate across a gap.

This ligase is from a Thermus species that was previously characterized as Thermus aquaticus HB8 (Taq DNA Ligase) and is currently classified as Thermus thermophilus HB8 (Tth DNA Ligase), and is commercially available under both names.

Properties:
Molecular Weight: 76.91 kDa
Optimum pH: 8.5 to 9.0
Optimum Mg²⁺ Concentration: 1mM
Co-factor: NAD⁺, optimum concentration is between 1-10mM
Ligation rate is unaffected by DTT (0 to 10mM) or Triton X-100 (0 to 1%)

Fidelity:

• Mismatched basepairs within 8 bases of the ligase junction on the 5' DNA strand decrease the rate of ligation⁽⁴⁾.
• Mismatched bases directly on either the 5' or 3' side of the ligation junction reduce ligation rate, however the ligation rate is more sensitive to 3'mismatches than to 5' mismatches⁽⁵⁾.

Purity:
Greater than 95% pure as determined by SDSPAGE. Tested for contaminating endonucleases, exonucleases, and ribonucleases.

Storage Buffer:
10mM Tris-HCl, pH 7.5, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Tween^®-20, 50% glycerol

Unit Definition:
One unit is the amount of enzyme required to ligate 1 pmol of sites in 1 hour at 45°C.

Concentration:
50 units/μl

Functionally Tested 10X Thermus DNA Ligase Reaction Buffer (1 ml included):
200mM Tris-HCl, pH 8.8, 500mM KCl, 100mM DTT, 10mM NAD⁺, 1% Tween^®-20, 1mM EDTA, 10mM MgCl₂.

References:

1. Barany, F. (1991) Proc. Natl. Acad. Sci. (USA) 88, 189-193.
2. Barany, F. and Gelfand, D. (1991) Gene 109, 1-11.
3. Pascal, JM (2008) Curr Opin Struct Biol. 18(1), 96-105.
4. Pritchard, C. and Southern, E. (1997) Nucleic Acids Res. 25, 3402-3407.
5. Luo, J., Bergstrom, D., and Barany, F. (1996) Nucleic Acids Res. 24, 3071-3078.

Storage:
Shipped on dry ice. Store at -20°C.