Transactivation capacity of WT and mutant p53 towards canonical and nncanonical REs.

WT p53 and several structural mutations were examined in three in vivo systems (haploid [1n] and diploid [2n] yeast and human SaOS2 cells) for transactivation from REs representing full, ?-sites and ?-sites. The ability of the p53, WT or mutant, to transactivate from a RE was measured with a luciferase assay and compared to the ability of WT p53 to transactivate from the p21-5�� RE at 0.008% and 0.024% galactose in 1n and 2n yeast, respectively, or compared to the pGL3 plasmid lacking the promoter REs in human cells. Relative transactivation by a p53 variant in comparison to WT p53 towards the p21-5�� RE is represented by +++ (75�C100%), ++ (25�C75%), + (7.5�C25%), +/? detectable but weak response, or ? (no detectable response). The R337C and R337H mutations are compromised for their ability to form tetramers (the equilibrium between dimers and tetramers is altered in comparison to WT p53) [86]. The L344A and L344P mutations result in p53 protein being present as a dimer and monomer, respectively. The ��368 mutation deletes the C-terminal basic domain required for structure-specific and non-sequence specific binding but has no effect on dimer or tetramer formation. ��a�� corresponds to significantly higher response at high galactose concentrations (0.128% and 2%). ��b�� indicates that the mutant was capable of transactivation from RE in an ADE2 plate assay at high galactose concentrations. ��d�� indicates that the dimer mutation N345S was also analyzed in the mammalian system and yielded the same results as L344A.