| The reversible modification of cellular proteins with adenosine diphosphoribose (ADP-ribose) polymers is closely linked to multiple mechanisms critical to the maintenance of genomic integrity. ADP-ribose polymer synthesis is catalyzed by poly(ADP-ribose) polymerases (PARPs) using nicotinamide adenine dinucleotide (NAD+) as substrate. PARP-1, the original and best understood PARP, has been shown to facilitate DNA repair or promote cellular death by apoptosis or necrosis. Tankyrase, another PARP but localized on telomeres, has been shown to regulate telomere length. The PARP family appears critical in maintaining genomic stability and has therefore been identified as a therapeutic target.; Poly(ADP-ribose) glycohydrolase (PARG) is the only cellular enzyme known to catalyze the hydrolysis of ADP-ribose polymers to free ADP-ribose. The rapid action of PARG and the transient nature of ADP-ribose polymers suggest a closely coordinated modus operandi between PARPs and PARG. Since PARG is potentially a more attractive target due to its low cellular levels and the uniqueness of its substrate, this project presents the structural features of PARG which allowed further insight into our understanding of this enzyme. Structural studies were made possible by producing recombinant PARG catalytic fragment (rPARG-CF) equivalent to PARG isolated from bovine thymus. This rPARG-CF was later purified to apparent homogeneity utilizing 8-(6-aminohexyl)aminoadenosine diphosphate (hydroxymethyl)pyrrolidinediol-agarose, an affinity resin containing ADP-HPD, the most potent and specific inhibitor of PARG to date. ADP-HPD was utilized as lead inhibitor in structure-activity relationship analyses which provided insight into the substrate structural requirements necessary for high affinity for the PARG active site. In addition, after mapping the active site region by photoaffinity labeling, the role of Tyr-796 as the substrate binding residue and the role of Glu-756/Glu-757 as the essential catalytic residues were confirmed by site-directed mutagenesis. As a result of these structural characterizations, a signature sequence, an active site model, and a catalytic mechanism of PARG were proposed. Finally, based in part on the structural data accumulated, techniques were established to study the effects of modulating intracellular levels of PARG in order to elucidate the biological consequences of this potentially novel therapeutic target in the future. |
