| Polymers of ADP-ribose (PAR) are rapidly synthesized by poly(ADP-ribose) polymerases (PARPs) and rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) following genotoxic stress. Since PAR metabolism plays an important role in cell fate determination following genotoxic stress, enzymes involved in PAR metabolism potentially represent promising therapeutic targets for modulating diseases of inappropriate cell proliferation or death. PARP-1 has been well validated and several PARP-1 inhibitors are currently being evaluated in clinical trials for cancer and ischemia treatment. In contrast, the biological function of PARG is still poorly understood. Due to low abundance of protein levels in mammalian cells and its unique substrate, PARG potentially represents another attractive target for pathological conditions mentioned above. PARG-Delta2,3 cells derived from homozygous PARG-Delta2,3 mice with targeted disruption of exons 2 and 3 of the PARG gene are used in this dissertation. The nuclear isoform PARG60 in PARG-Delta2,3 cells lacks the putative regulatory domain A compared to the nuclear isoform PARG110 in wild type cells.; We report in this dissertation that PARG-Delta2,3 cells accumulate less PAR in spite of more rapid depletion of NAD following treatment with N-methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). The estimation of PARP and PARG activity in intact cells shows increased activity of both enzymes in PARG-Delta2,3 cells following MNNG treatment, indicating the important role of domain A in the regulation of PARG and PARP activity under these conditions. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, decreased H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. The altered PAR metabolism and defective cellular responses related to DNA repair in PARG-Delta2,3 cells may contribute to increased sensitivity of these cells to MNNG. Studies presented in this dissertation clearly demonstrate the important role of PARG110 in PAR metabolism and cellular responses to genotoxic stress, and thus provide supportive data for the validation of PARG as a promising potential therapeutic target. |
