Characterization of an ATP-dependent DNA ligase from the the thermophilic archaeon Methanobacterium thermoautotrophicum Journal Article

   


Authors: Sriskanda, V.; Kelman, Z.; Hurwitz, J.; Shuman, S.
Article Title: Characterization of an ATP-dependent DNA ligase from the the thermophilic archaeon Methanobacterium thermoautotrophicum
Abstract: 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 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.
Keywords: controlled study; mutation; nonhuman; enzyme degradation; protein degradation; calcium; in vitro study; enzyme activity; bacteria (microorganisms); amino acid sequence; molecular sequence data; kinetics; enzyme analysis; recombinant proteins; substrate specificity; temperature; amino acid; binding sites; adenosine triphosphate; cobalt; protein folding; polydeoxyribonucleotide synthase; nick end labeling; guanosine triphosphate; bacterial dna; enzyme purification; centrifugation, density gradient; lysine; archaea; hydrogen-ion concentration; asparagine; enzyme synthesis; nicotinamide adenine dinucleotide; archaeal proteins; dna ligases; enzyme active site; divalent cation; adenosine phosphate; endopeptidases; nucleotidyltransferase; cations, divalent; magnesium; bacterial mutation; glycerol; bacterial enzyme; prokaryota; manganese; salts; cytidine triphosphate; deoxyadenosine triphosphate; thermophilic bacterium; uridine triphosphate; methanobacterium thermoautotrophicum; methanobacterium; phocidae; priority journal; article; methanothermobacter thermautotrophicus
Journal Title: Nucleic Acids Research
Volume: 28
Issue: 11
ISSN: 0305-1048
Publisher: Oxford University Press  
Date Published: 2000-06-01
Start Page: 2221
End Page: 2228
Language: English
PUBMED: 10871342
PROVIDER: scopus
PMCID: PMC102631
DOI: 10.1093/nar/28.11.2221
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-0034209827&partnerID=40&md5=ca25a0931cdd709b225315cbc56b2a1e
Notes: Export Date: 18 November 2015 -- Source: Scopus
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MSK Authors
  1. Verl S Sriskanda
    13 Sriskanda
  2. Jerard Hurwitz
    206 Hurwitz
  3. Stewart H Shuman
    546 Shuman
  4. Zvi Kelman
    17 Kelman
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