p53 mutants exhibiting enhanced transcriptional activation and altered promoter selectivity are revealed using a sensitive, yeast-based functional assay

Abstract

Changes in promoter speci®city and binding anity that may be associated with p53 mutations or post-translational modi®cations are useful in understanding p53 structure/ function relationships and categorizing tumor mutations. We have exploited variable expression of human p53 in yeast to identify mutants with novel phenotypes that would correspond to altered promoter selectivity and anity. The p53 cDNA regions coding for the DNA binding and tetramerization domains were subjected to random PCR mutagenesis and were cloned directly by recombination in yeast into a vector with a GAL1 promoter whose level of expression could be easily varied. p53 variants exhibiting higher than wild type levels of transactivation (supertrans) for the RGC responsive element were identi®ed at low level of p53 protein expression. All the p53 mutants obtained with this screen were located in the DNA binding domain. Two out of 17 supertrans mutants have been found in tumors. Six mutations were in the L1 loop region between amino acids 115 and 124. The transactivation potential of a panel of supertrans p53 mutants on dierent promoters was evaluated using the p53 responsive elements, RGC, PIG3, p21 and bax. Although all mutants retained some activity with all promoters, we found dierent patterns of induction based on strength and promoter speci®city. In particular none of the mutants was supertrans for the p21 responsive element. Interestingly, further analysis in yeast showed that the transactivation function could be retained even in the presence of dominant-negative p53 tumor mutations that could inhibit wild type p53. Five mutants were also characterized in human cells in terms of growth suppression and transactivation of various promoters. These novel supertrans p53 mutants may be useful in studies aimed at dissecting p53 downstream pathways, understanding speci®c interactions between p53 and the DNA, and could replace wild type p53 in cancer gene therapy protocols. The approach may also prove useful in identifying p53 tumor mutations. Oncogene (2001) 20, 501 ± 513.

Comparison of the protein–protein interfaces in the p53–DNA crystal structures: Towards elucidation of the biological interface

“…The large conformational change of loop1 observed in our simulations provides insight into p53-DNA binding, which has been difficult to observe from the crystal structure alone. Mutations in the loop1 region enhance specific p53-DNA binding (32), probably because of the stabilization of loop1. The recently solved crystal structure of Cep-1, the human p53 Caenorhabditis elegans ortholog (33) also highlights the need to understand the function of the loop1.…”

Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity

“…Based on Western blot analysis (ref. 21 and unpublished results) the differences in mutant p53 activities cannot be ascribed to intrinsic differences in stability. Along this line, the present results that demonstrate dramatic shifts in the spectra and strength of transactivation at many REs with single amino acid changes in p53 suggest that simple structural and energy models for predicting binding efficiency of regulatory proteins do not explain the strength of binding to REs (9,18).…”

“…Mutants 121F through 124Y and 288K were identified in two separate screens for p53 alleles that could lead to enhanced transactivation (supertrans) at the RGC or p21-5Ј RE (ref. 21 and unpublished results). Mutant 288D was included as a comparison with 288K.…”

“…5, which is published as supporting information on the PNAS web site). The opportunity to vary the p53 expression levels enabled us to identify mutations that had altered abilities to transactivate from some of these REs (20,21).…”

Interactions of mutant p53 with DNA: guilt by association

“…Since these seminal findings, the understanding of the biology of mutp53 proteins has advanced enormously, the quintessence being the realization that mutp53 proteins are extremely heterogeneous in their structural and possibly also in their biochemical properties (see Joerger and Fersht, 2007, this issue; reviewed by Sigal and Rotter, 2000). Underscoring the inequality of structural defects in the core domains of mutp53 proteins, the major activity of the core domain, SSDB, is impaired in individual mutp53 proteins to different degrees, with the severity of SSDB impairment spanning a broad range (see Joerger and Fersht, 2007;Menendez et al, 2007, this issue;Inga et al, 2001;Ang et al, 2006). However, except for the residual SSDB activity retained by the core domain of some mutp53 proteins, the principles of DNA recognition by mutp53 proteins and the role of mutp53 DNA binding for the oncogenic activities associated with some mutp53 proteins remains far from being clear.…”