Figure 6.
Formation of PAR–DNA adducts in nuclear extracts from HeLa cells. Twenty nanomolar 5′-[32P]labeled 7–13-3′tP-Db32gap (A) or 10-Db32nick (B) Dbait molecules were incubated with the indicated amount of HeLa extracts, 200 nM olaparib, 180 nM PARP1 and 40 nM PARP2 or 50 nM PARP3 under standard reaction conditions for a PARP-dependent DNA ADP-ribosylation assay. Incubation periods were 10 min for extracts, 30 min for PARP1 and PARP2, and 2 min for PARP3. Lanes 6 and 9 show repeats of the experiments in lanes 5 and 8, respectively. The reaction products were analyzed by denaturing PAGE. For details, see ‘Materials and Methods’ section and Supplementary Figure S12.