Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum.
We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid MTH: ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K(m) 1.1 microM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD(+) cannot. MTH: ligase activity is thermophilic in vitro, with optimal nick-joining at 60 degrees C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase-adenylate intermediate (step 1) and hence cannot seal a 3'-OH/5'-PO(4) nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase-adenylate formation, but defective in activating the nick via formation of the DNA-adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA-adenylate, could be elicited for the wild-type MTH: ligase by inclusion of calcium as the divalent cation cofactor. MTH: ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that MTH: ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.
Sriskanda
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V
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Kelman
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Z
Hurwitz
J
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Shuman
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D019843:Archaeal Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D012492:Salts; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D011088:DNA Ligases