Figure 6.

PARylation is independent of cross-linking of PARP-1. (a) A reaction mixture (40 μl) containing purified PARP-1 and ³²P-labeled AP site-containing DNA was incubated on ice without NaBH₄ under similar reaction conditions as in Figure 1. After this incubation, the reaction mixture was divided into four equal portions and each reaction mixture was supplemented as follows: lane 1, buffer; lane 2, 100 μM NAD; lane 3, 100 μM NAD and 100 ng PARG; and lane 4, 100 ng PARG. All the reaction mixtures also contained 7.5 mM MgCl₂. Incubation was 15 min at 30°C. (b) A reaction mixture (20 μl) containing purified PARP-1, ³²P-labeled AP site-containing DNA, 100 100 μM NAD and 7.5 mM MgCl₂ was incubated as in panel (a). After this incubation, the reaction mixture was divided into two equal portions and each reaction mixture was supplemented as follows: lane 1, buffer, and lane 2, 100 ng PARG. Incubation was 15 min at 30°C. (c) and (d) The experiments were repeated as described in panels (a) and (b), except the DNA substrate had a nick and was ³²P-labeled in the nick as depicted above the phosphorimage. The migration positions of cross-linked PARP-1 (XL-PARP-1) and PARylated PARP-1 are indicated.