title: Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.
abstract: Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sekiguchi
authors.forename: S
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D011088:DNA Ligases / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D010710:Phosphates; D000249:Adenosine Monophosphate; D004254:DNA Nucleotidyltransferases; C058134:vaccinia virus nicking-joining enzyme; D004264:DNA Topoisomerases, Type I; D011088:DNA Ligases