Figure 4.

Proteolysis of cross-linkined polypetides I and II with trypsin. (a) Phosphorimage illustrating the proteolysis of the PARP-1-DNA cross-linked complex with trypsin. Purified polypeptides I and II and ³²P-labeled AP site DNA were incubated on ice without NaBH₄ under similar reaction conditions as in Figure 1 (lanes 1–4). After the intrinsic cross-linking, the pH of the reaction mixture was adjusted to 8.0 by adding 1 M Tris-HCl, pH 8.0, to a final concentration of 100 mM. The covalently cross-linked complex was then treated with trypsin at a 1:5 ratio of trypsin to complex (w/w), and the mixture was incubated at 25ºC for 20 min. The proteolysis products were analyzed as in Figure 1. Cross-linked polypeptide III was used as reference (lane 5). (b) Photograph of the Coomassie blue-stained gel in panel (a). Schematic representation of a 5′-end ³²P-labeled 34 bp DNA with an incised natural AP site is shown above the phosphorimage in (a).