Figure 2.

Role of cooperativity for DNA binding of p53 in the human genome. (A) Schematic representation of the dimerization patterns of wild-type p53 and the H1 helix mutants used in this study. The small insert shows the 3D structure of the double salt bridge in the wild-type molecule. To disrupt the intradimer interface we introduced modest charge-neutralizing (E180→L “LR” and R181→L “EL”) and more severe charge-inverting (E180→R “RR” and R181→E “EE”) mutations into the H1 helix of the full-length p53 molecule. The short names denote the amino acid sequence at positions 180 and 181 in the mutant proteins, e.g., “ER” for E180, R181 in the wild-type. To assure that functional defects are truly due to defective core domain interactions and are not caused by structural misfolding of the core domain or disturbed interaction with other cellular proteins, we also introduced the two most severe mutations E180R and R181E together into a single p53 molecule (double mutant E180R, R181E “RE”) and used the two complementing mutants “EE” and “RR” in functional rescue studies. (B) p53 DNA binding cooperativity determines the number of binding sites in the genome. The number of binding sites was estimated by bioinformatic analysis combining ChIP-chip results with experimental validation rates determined by ChIP-qPCR.⁹ (C) De novo motif discovery in validated common EL/RE and RE-only binding sequences. Twenty-meric and decameric consensus motifs are shown for comparison. (D and E) Frequency and average motif scores of the TRANSFAC motifs V$P53_01 (full site), V$P53_02 (half-site) and V$E2F_01 (E2F site as a control) in validated common EL/RE and RE-only binding sequences. Results are presented as the mean ± SD. (F) Distribution of spacer lengths in validated common EL/RE and RE-only binding sequences as determined by the spacer-tolerant p53MH algorithm.