Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon "Ferroplasma acidarmanus" Fer1.
Analysis of the genome of "Ferroplasma acidarmanus" Fer1, an archaeon that is an extreme acidophile, identified an open reading frame encoding a putative ATP-dependent DNA ligase, which we termed FaLig. The deduced amino acid sequence of FaLig contains 595 amino acids, with a predicted molecular mass of 67.8 kDa. "F. acidarmanus" Fer1 is classified as a Euryarchaeote, but phylogenetic analysis using amino acid sequences showed that FaLig is more similar to DNA ligases from Crenarchaeota, suggesting that lateral transfer of these genes has occurred among archaea. The gene sequence encoding FaLig was cloned into a bacterial expression vector harbouring an upstream His-tag to aid purification. Conditions for expression and purification from Escherichia coli were identified and recombinant FaLig was confirmed to be an ATP-dependent DNA ligase. Optimal conditions for nick-joining by the protein were pH 6-7, 0.5 mM ATP, in the presence of either Mg(2+) or Mn(2+). Using a range of nicked, double-stranded nucleic acids, ligation was detected with the same substrates as previously determined for other DNA ligases. Although FaLig is the DNA ligase from one of the most extreme acidophilic organism yet studied, this characterization suggests that its biochemical mechanism is analogous to that of enzymes from other cellular systems.
Jackson
Brian R
BR
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.
Noble
Catherine
C
Lavesa-Curto
Manuel
M
Bond
Philip L
PL
Bowater
Richard P
RP
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020745:Genome, Archaeal / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny
D019843:Archaeal Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium