title: Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.
abstract: In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F) is described. We show that the kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.
authors.affiliation: Ecole Supérieure de Biotechnologie de Strasbourg, UPR 9003 du Centre National de la Recherche Scientifique, Illkirch Graffenstaden, France.
authors.forename: E A
authors.initials: EA
authors.lastname: Miranda
authors.forename: F
authors.initials: F
authors.lastname: Dantzer
authors.forename: M
authors.initials: M
authors.lastname: O'Farrell
authors.forename: G
authors.initials: G
authors.lastname: de Murcia
authors.forename: J M
authors.initials: JM
authors.lastname: de Murcia
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000187:drug effects; D004249:DNA Damage; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D009243:NAD / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D006898:Hydroxylamines; D009243:NAD; D019811:Hydroxylamine; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases