FIG 6.

Target DNA binding affinity of recombinant wild-type p53 and mutant p53^Y220C. (A) EMSA with purified wild-type p53 and mutant p53^Y220C shows similar DNA binding affinity for the target DNA. A fixed concentration of ³²P-labeled 30-bp WAF-side DNA was incubated with increasing concentrations of purified recombinant wild-type p53 (left) or mutant p53^Y220C (right) at 37°C. The mixture was subjected to EMSA. The bound WAF-side DNA level versus the tetrameric p53 concentration was plotted using GraphPad, and the dissociation constant was determined using a nonlinear regression curve fit for one-site binding of DNA with tetrameric p53. (B) ITC of 30-bp WAF-side DNA binding to the wild-type p53 (left) and mutant p53^Y220C (right) shows similar binding affinity. A syringe of ITC containing WAF-side DNA was titrated into a cell containing purified wild-type or mutant p53 in ITC buffer containing 20 mM sodium phosphate buffer, pH 7.8, and 150 mM NaCl at 37°C. (Top) The calorimetric data of titration of p53 with DNA as a function of time. (Bottom) The integrated heat per injection versus the molar ratio of tetrameric p53 to DNA. The graph corresponds to the best fit of the experimental data to the one-site model, providing a dissociation constant (37) of 5.5 nM for the wild-type p53 and 7.2 nM for the mutant p53^Y220C. (C) Inhibition of DNA binding activity of wild-type p53 by recombinant FBP1. The purified recombinant p53 (7 nM) was incubated with increasing concentrations of FBP1 (25 to 100 nM) in a final volume of 20 μl. After 30 min of incubation at room temperature, a fixed concentration of ³²P-labeled 30-bp WAF-side DNA (40,000 cpm) was added; the mixture was incubated for 30 min at 37°C. The p53-DNA complexes were resolved by EMSA. Lane 1, p53 binding to DNA in the absence of FBP1; lanes 2 through 5, p53 binding to DNA in the presence of 25, 50, 75, and 100 nM recombinant FBP1. (D) Binding of FBP1 alone to ³²P-labeled WAF-side DNA is not significant. Lanes 1 through 4, increasing concentrations of FBP1 (25 to 100 nM) were incubated at room temperature with ³²P-labeled WAF-side DNA (40,000 cpm) in a final volume of 20 μl for 30 min. The bound and unbound DNA were resolved by EMSA. (E) EMSA with a nontarget ³²P-labeled 30-bp HIV-1 U5 PBS DNA was used to determine the target specificity of wild-type p53 and mutant p53^Y220C.