Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using λ DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx.volcanii cells.LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels.DNA ligases play key roles in all forms of cellular life [1]. Two families of DNA ligase, differing in their cofactor specificity, are characteristic of the two major branches of evolution, the eubacteria and the eukarya/archaea. NAD+-dependent DNA ligases (EC 6.5.1.2) are encoded predominantly by eubacteria [2] but also by certain eukaryotic viruses, such as the entomopoxviruses [3] and mimiviruses [4], and by some bacteriophage [5,6]. In contrast, ATP-dependent DNA ligases (EC 6.5.1.1) are characteristic of eukaryotic and archaeal cells but are also found encoded by certain eukaryotic viruses, bacteriophage and eubacteria [7]. The mechanism of DNA ligation is similar for both types of enzyme [1]. In the first step, attack on the α-phosphorus of either NAD+ or ATP by the enzyme results in formation of an enzyme-adenylate intermediate (AMP is covalently linked to a lysine residue) and release of either NMP or pyrophosphate. In the second step, the AMP moiety is transferred to the 5' end of the nicked DNA strand to form a DNA-adenylate complex.