Nuclear ADP-ribose polymers bound to proteins modulate chromatin structure and function in higher eucaryotes. These polymers are synthesized by poly (ADP-ribose) polymerase (PARP), a chromatin-bound enzyme [EC 2.5.230], that utilizes BNAD as the ADP-ribose substrate and is activated by DNA-strand breaks. This protein of 1014 amino acids in size utilizes DNA-binding proteins as (ADP-ribose)n acceptors. It also catalyzes the ADP-ribose chain initiation, elongation, and branching reactions of polymer synthesis via homodimerization (automodification) and heterodimerization with protein acceptors (heteromodification). Protein-poly(ADP-ribosyl)ation appears to be required for the accurate rejoining of nicks and breaks on DNA that occur during DNA-replication, gene expression, and DNA-excision repair. The gene and full cDNA sequence of PARP have recently become available. The amino acid sequence of PARP reveals a tripartite domain structure. The function of these domains is to bind DNA (amino-terminus), automodification sites (central region), and substrate binding (carboxy-terminus). However, the molecular cooperation of the various domains in ADP-ribose polymer synthesis and the enzyme mechanisms of PARP remain to be established.
The specific aims of this proposal are to: 1) dissect the synthesis of poly (ADP-ribose) into individual reactions of initiation, elongation, and branching in the automodification reaction; 2) characterize the elongation, and branching steps with mono(ADP-ribose)-histone peptide domains as the acceptor(s) (heterologous poly(ADP-ribosyl)ation); 3) determine the kinetic mechanics and the acid-based chemistry of the ADP- ribose elongation reaction catalyzed by PARP; and 4) utilize site- directed mutants (SDM's) of the PARP gene to determine the catalytic role of specific amino acid groups of the enzyme. This research project will also identify and characterize the mechanism(s) that modulate(s) the multiple enzymatic activities of protein-protein interactions of this enzyme with itself (intermolecular dimeric automodification) as well as other chromatin components (heterodimerization).
In specific aim #1, specific assays for each reaction will be developed. The initiation reaction will be characterized at nanomolar concentrations of NAD in the presence of agmatine-(ADP-ribose) to specifically inhibit chain elongation. The elongation reaction will be performed with specific automodification peptide fragments covalently bound to monomeric ADP- ribose(mock initiation agmatine-ADP-ribose). This inhibitor blocks the enzyme binding site for the ADP-ribose elongation acceptor which forces PARP to catalyze branching. Similar experiments will be performed in the presence of mono(ADP-ribosyl)ated-peptides containing the acceptor domains of histone H1 and/or H2b in specific aims #2. Inhibition studies with benzamide(s), Agmatine-(ADP-ribose) and 2'dNAD will also be carried out to achieve specific aim #3.
In specific aim#4, SDM's of PARP will be utilized to confirm the catalytic role of the amino acid residues involved in the acid-based chemistry of ADP-ribose polymer synthesis.
| Pacheco-Rodriguez, G; Alvarez-Gonzalez, R (1997) The minimum size that a protein-free ADP-ribose chain requires to precipitate in 20% (w/v) trichloroacetic acid is 14 units. Anal Biochem 247:452-5 |
