Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae.
We report that Haemophilus influenzae encodes a 268 amino acid ATP-dependent DNA ligase. The specificity of Haemophilus DNA ligase was investigated using recombinant protein produced in Escherichia coli. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km = 0.2 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. Haemophilus ligase reacted with ATP in the absence of DNA substrate to form a covalent ligase-adenylate intermediate. This nucleotidyl transferase reaction required a divalent cation and was specific for ATP. The Haemophilus enzyme is the first example of an ATP-dependent DNA ligase encoded by a eubacterial genome. It is also the smallest member of the covalent nucleotidyl transferase superfamily, which includes the bacteriophage and eukaryotic ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes.
Cheng
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Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021 USA.
Shuman
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D000225:Adenine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004926:Escherichia coli; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium; D000225:Adenine