Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans.
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.
Rolland
Jean-luc
JL
Ifremer, Direction des Ressources Vivantes, France.
Gueguen
Yannick
Y
Persillon
Cècile
C
Masson
Jean-Michel
JM
Dietrich
Jacques
J
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
D003067:Coenzymes; D019641:DNA, Archaeal; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D009243:NAD; D000255:Adenosine Triphosphate; D011088:DNA Ligases