FIG. 5.
Purification and enzymatic activities of PARP-1 mutants. The PARP-1 mutants shown in Fig. 4A and 6A were expressed in E. coli and purified by nickel-NTA affinity chromatography (see Materials and Methods). (A) Purified wild-type and mutant PARP-1 proteins were analyzed by SDS-PAGE with staining by Coomassie brilliant blue R-250. Molecular mass markers are shown. (B) Automodification activities of wild-type and mutant PARP-1 proteins. The automodification activities of the indicated PARP-1 proteins were determined in the presence of [32P]NAD+, as described in Materials and Methods. Sheared DNA was added as an allosteric activator of PARP-1. The samples were analyzed by SDS-PAGE with detection of the 32P signal by phosphorimager analysis.