Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching.
Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.
Rolli
V
V
Ecole Supérieure de Biotechnologie de Strasbourg, UPR A9003 du CNRS, Illkirch-Graffenstaden, France.
O'Farrell
M
M
Ménissier-de Murcia
J
J
de Murcia
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics*; D002384:Catalysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011108:Polymers / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D000596:Amino Acids; D011108:Polymers; D011065:Poly(ADP-ribose) Polymerases