FIG. 7.

cdc2 inhibition of TFIIH kinase activity is reversed by the cyclin-dependent kinase inhibitor p21. (A) Affinity-purified cdc2/GST-cyclin B was incubated with the indicated concentrations of p21, and these mixtures were then used in kinase reactions with histone H1 as a substrate. The position of ³²P-labeled histone H1 is indicated. The positions of molecular mass markers (in kilodaltons) are indicated to the left. (B) HeLa cell nuclear extract was incubated with (+) or without (−) 1 μM p21, and antibody to p62 (αp62) was used to immunopurify TFIIH from the treated or untreated extract. These immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of ³²P-labeled GST-CTD_o and GST-CTD_a are shown. (C) Affinity-purified cdc2 was incubated without or with p21 at various concentrations as indicated. Next, HeLa cell extract was incubated with this p21-treated cdc2. Antibody to p62 was used to immunopurify TFIIH from the cdc2-treated HeLa cell extracts. The TFIIH immunoprecipitates were then used in kinase assays to phosphorylate GST-CTD. The positions of ³²P-labeled GST-CTD_o and GST-CTD_a are shown.