title: 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.
abstract: BACKGROUND
  DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx. volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx. volcanii ATP-dependent DNA ligase protein LigA.
  
  
  RESULTS
  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 lambda 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Molecular Biology and Physiology, University of Copenhagen, Sølvgade 83H, 1307 Copenhagen, Denmark. poidevin_titia@yahoo.fr <poidevin_titia@yahoo.fr>
authors.forename: Laetitia
authors.initials: L
authors.lastname: Poidevin
authors.forename: Stuart A
authors.initials: SA
authors.lastname: MacNeill
mesh_terms: D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019622:Haloferax volcanii / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D011189:Potassium Chloride / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts
chemical_list: D011994:Recombinant Proteins; D012492:Salts; D011189:Potassium Chloride; D011088:DNA Ligases; C120640:DNA ligase (NAD)