Fig. 5. PARP activation by UVB-irradiated closed circular plasmid DNA. (A) PARP activation in vitro with UVB-irradiated plasmid. Purified PARP was activated with control, UVB (3.2 kJ/m²)- or UVB + T4 EndoV-treated circular plasmid DNA in the presence of 1.2 µM ³²P-NAD. Aliquots were resolved on SDS-PAGE prior to autoradiography. Results of one of four experiments with identical results is shown. (B) Quantification of PARP activation in the in vitro assays. PARP was reacted with three plasmids, as described above, along with activated, i.e. extensively nicked DNA. Reaction was terminated at 30 minutes and ³²P-pADPr-modified PARP was TCA precipitated and counted. This experiment was carried out four times with each assay conducted in triplicate and data are presented as mean±s.e.m. of 12 observations. (C) In vitro PARP activation assay with non-isotopic NAD. In vitro PARP activation assay with control and UVB-irradiated plasmid DNA was carried out with 200 µM non-isotopic NAD for 60 minutes. The unmodified PARP and pADPr-modified PARP were detected by immunoblotting with antibodies specific for PARP and pADPr. Data represent four experiments with identical results.