title: Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.
abstract: Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis. Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA. Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes. In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.
authors.affiliation: Institut de Biologie Moléculaire et cellulaire du Centre National de la Recherche Scientifique, Laboratoires de Biochimie, Strasbourg, France.
authors.forename: F
authors.initials: F
authors.lastname: Simonin
authors.forename: J
authors.initials: J
authors.lastname: Ménissier-de Murcia
authors.forename: O
authors.initials: O
authors.lastname: Poch
authors.forename: S
authors.initials: S
authors.lastname: Muller
authors.forename: G
authors.initials: G
authors.lastname: Gradwohl
authors.forename: M
authors.initials: M
authors.lastname: Molinete
authors.forename: C
authors.initials: C
authors.lastname: Penning
authors.forename: G
authors.initials: G
authors.lastname: Keith
authors.forename: G
authors.initials: G
authors.lastname: de Murcia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013950:Thymus Gland / Q000201:enzymology; D014158:Transcription, Genetic
chemical_list: D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; D008239:Lysine