title: Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans.
abstract: A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.
authors.affiliation: Ifremer, Direction des Ressources Vivantes, France.
authors.forename: Jean-luc
authors.initials: JL
authors.lastname: Rolland
authors.forename: Yannick
authors.initials: Y
authors.lastname: Gueguen
authors.forename: Cècile
authors.initials: C
authors.lastname: Persillon
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Masson
authors.forename: Jacques
authors.initials: J
authors.lastname: Dietrich
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019641:DNA, Archaeal / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D003067:Coenzymes; D019641:DNA, Archaeal; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D009243:NAD; D000255:Adenosine Triphosphate; D011088:DNA Ligases