Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Abstract

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65-70°C and retains its DNA ligation activity even after heated at 100°C for 2h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3' to the nick suppress Tba ligase activity more than those at the first position 5' to the nick. The enzyme also discriminates more effectively mismatches at 3' to the nick than those at 5' to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.