Nucleic Acids Research, 2000, Vol. 28, No. 11 2221-2228
© 2000 Oxford University Press
Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA
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 (Km 1.1 µM). 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°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'-PO4 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.
* To whom correspondence should be addressed. Tel: +1 212 639 7145; Fax: +1 212 717 3623; Email: s-shuman@ski.mskcc.org
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