Mechanism of κB DNA binding by Rel/NF-κB dimers*

The DNA binding of three different NF-κB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-κB p50/p65 heterodimer is shown here to bind the κB DNA target site of the immunoglobulin κ enhancer (Ig-κB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-κB complex. The heterodimer binds to the Ig-κB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-κB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 °C and 42 °C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-κB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.