Figure 2.
Destabilization of the protein–DNA complex by 19S is dependent on ATP and the activation domain. (A) The influence of ATP and the activation domain in protein–DNA complex destabilization by 19S. Isolation and destabilization of the indicated protein–DNA complex by 19S was carried out in the presence or absence of ATP as described in Figure 1B. (B) ATP hydrolysis is required for efficient destabilization of the activator–DNA complex by 26S proteasome. Destabilization of the activator–DNA complex by 26S proteasome was analyzed in the presence of a 10-fold excess of specific competitor DNA as described in Figure 1B, except that 500 μM of ATP and the indicated ATP analogs were added. (C) Destabilization of the GST-Pho4p/DNA complex by the proteasome. Destabilization of Pho4p–DNA complexes by 26S proteasome was performed as described in Figure 1B, except that the DNA template contained two Pho4-binding sites and the proteasome was preincubated with MG132.