| In the Myc/Max/Mad network, Max and Myc form a heterodimer that has strong oncogenic potential, while Mad and Max form a heterodimer that acts as a tumor suppressor. The mechanism of the transcription function of Myc/Max/Mad network is directly related to the interaction between these protein dimers and their DNA recognition site, a DNA E-box element, CACGTG. The quantitative data on these interactions does not clearly illustrate the binding mechanism. Fluorescence anisotropy has been used in our experimental approach to measure protein-protein and protein-DNA binding affinity. The enthalpy and entropy of these interactions were determined from the temperature dependence. The Max homodimer binds to the DNA with higher affinity than Myc/Max or Mad/Max heterodimers, but has less specificity for the E-box and flanking sequence recognition. The overall DeltaG o for formation of the protein dimer-DNA complex was almost 4 kJ less favorable for Mad-Max-DNA formation at 20°C compared to Max2-DNA or Myc-Max-DNA, which were essentially the same. However, at 37°C Mad-Max-DNA and Myc-Max-DNA have essentially the same DeltaGo while Max2-DNA was about 7 kJ less stable. The enthalpy values for Max2-DNA formation were significantly higher than for either of the heterodimer-DNA interactions. The largest contribution was assigned to the protein-DNA interaction rather than the protein-protein interaction. These results suggest that the formation of heterodimers between Max and other proteins are a more important function of Max than the selection of DNA sequences. The fluorescence anisotropy measurement also suggests that for the unphosphorylated Max, the dimerization and protein-DNA interaction pathway might be favorable to the formation of Max monomer-DNA complex, instead of the formation of Max heterodimer. This result is consistent with the idea, that the Max phosphorylation by Casein Kinase II is the core upstream regulation of the entire Myc/Max/Mad network. This regulation affects the Max binding affinity with E-Box DNA. In Addition, the fluorescence resonance energy transferring (FRET) experiment on the protein-DNA interaction did not show significant difference between Myc/Max and Mad/Max heterodimer, interacting with E-box oligonucleotides. It might suggest that those heterodimers bind to the DNA with similar orientations, or if the orientation were different in vivo, there has to be some other factors involved. |
