Proteins are reversibly modified with ADP-ribose polymers by the combined action of poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). The activation of PARP-1 and PARP-2 following genotoxic stress leads to rapid polymer synthesis, rapid polymer turnover as a result of PARG activity, and it can lead to depletion of cellular NAD. DNA break-activated PARPs are promising targets for the modulation of cellular responses to genotoxic stress and the closely coordinated activities of PARPs and PARG indicates that PARG may also be a good therapeutic target. We have approached target validation of PARG using several approaches. The cloning and expression of the cDNA coding for bovine [1] and human [2] PARG has allowed structural characterizations that make possible molecular genetic approaches to PARG modulation. Photoaffinity labeling and site-directed mutagenesis has led to the identification of three acidic residues essential for catalytic activity, allowing the expression of dominant negative forms of PARG. The cDNA sequence has allowed the design of RNA interference approaches for modulating PARG activity. The discovery of a sensitive and selective inhibitor of PARG [3], ADP-HPD, has led to the synthesis of a cell permeable inhibitor that can be used to modulate PARG activity in cultured cells. This presentation will compare the efficiency of the different approaches in modulating PARG activity and the effects of PARG modulation on NAD and ADP-ribose polymer metabolism and biological responses to genotoxic stress. Supported by grants from the NIH (CA-43894) and Niadyne, Inc. RGM was supported by NIH Cancer Biology Training Grant CA-09213.References: 1.Lin et al: J Biol Chem, 1997; 272: 11895-11901 2.Meyer et al: 2003, submitted 3.Slama et al: J Med Chem, 1995; 38: 389-393

Keywords: Poly(ADP-ribose) glycohydrolase, PARG, therapeutic targets, genotoxic stress