title: The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity.
abstract: Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
authors.forename: V
authors.initials: V
authors.lastname: Schreiber
authors.forename: M
authors.initials: M
authors.lastname: Molinete
authors.forename: H
authors.initials: H
authors.lastname: Boeuf
authors.forename: G
authors.initials: G
authors.lastname: de Murcia
authors.forename: J
authors.initials: J
authors.lastname: Ménissier-de Murcia
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002384:Catalysis; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D016335:Zinc Fingers; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011065:Poly(ADP-ribose) Polymerases; D001616:beta-Galactosidase