A novel resource centre for TP53 mutations and mutants has been developed (http://p53.fr). TP53 gene dysfunction can be found in the majority of human cancer types. The potential use of TP53 mutation as a biomarker for clinical studies or exposome analysis has led to the publication of thousands of reports describing the TP53 gene status in >10 000 tumours. The UMD TP53 mutation database was created in 1990 and has been regularly updated. The 2012 release of the database has been carefully curated, and all suspicious reports have been eliminated. It is available either as a flat file that can be easily manipulated or as novel multi-platform analytical software that has been designed to analyse various aspects of TP53 mutations. Several tools to ascertain TP53 mutations are also available for download. We have developed TP53MULTLoad, a manually curated database providing comprehensive details on the properties of 2549 missense TP53 mutants. More than 100 000 entries have been arranged in 39 different activity fields, such as change of transactivation on various promoters, apoptosis or growth arrest. For several hot spot mutants, multiple gain of function activities are also included. The database can be easily browsed via a graphical user interface.

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The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.T he p73 transcription factor that belongs to the p53 protein family and participates in pheromonal sensory, chromosome stability, neurogenesis, inflammation, and osteoblastic differentiation pathways (1, 2). In contrast to p53, p73 is mutated in less than 0.5% of human tumors (3); however, it also participates in p53-dependent and independent pathways, showing oncogenic and tumor suppressor functions (4, 5). These dual opposite activities are due to the presence of two promoters which results in the expression of two main isoforms, TAp73 and ΔNp73 (6).How the members of the p53 protein family trigger different cellular responses still remains an open question. Overall, p73 and p63 can bind to the same p53 response elements (REs), but the activated pathways are different (7,8). There is some redundancy in the activation of stress pathways by the three members of the p53 protein family, but, at the same time, over 100 genes regulated by p73 and p63 are not activated by p53 (9, 10). Like p53, p73 also binds to a 20-bp RE, comprising two half-site decamers in direct orientation that follow a 5′-Pur1-Pur2-Pur3-Cyt4-Ade5/Thy5-Ade6/Thy6-Gua7-Pyr8-Pyr9-Pyr10-3′ consensus sequence (10, 11). Half of the known p53 REs do not have any insertion between the two half-sites and spacers larger than 3 bp are rare, particularly among sites that are transcriptionally activated (12-14). In the case of p53 repressed genes, the cis-element code is poorly defined, but based on a limited number of examples, spacer length appears to be more uniformly distributed and targets have no preference for 0-bp spacers (12).Human p73α is a 636 amino acid protein with a tripartite domain organization similar to its close homolog, p63, and to the shorter 393 amino acid long p53 protein. Members of the p53 family have a disordered N-terminal transactivation domain, a central immunoglobulin-like DNA-binding domain (DBD), and a C terminus that starts with a domain that prom...

BackgroundThe p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4.Methodology/Principal FindingsGiven the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i) variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.Conclusions/SignificanceWe found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N 3 -methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT 3 TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A 600 -602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n ‫؍‬ 9, p < 10 ؊6 , Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

Germline TP53 mutations result in cancer proneness syndromes known as Li-Fraumeni, Li-Fraumeni-like, and nonsyndromic predisposition with or without family history. To explore genotype/phenotype associations, we previously adopted a functional classification of all germline TP53 mutant alleles based on transactivation. Severe deficiency (SD) alleles were associated with more severe cancer proneness syndromes, and a larger number of tumors, compared with partial deficiency (PD) alleles. Because mutant p53 can exert dominant-negative (DN) effects, we addressed the relationship between DN and clinical manifestations. We reasoned that DN effects might be stronger in familial cancer cases associated with germline TP53 mutations, where mutant alleles coexist with the wild-type allele since conception. We examined 104 p53 mutant alleles with single amino acid substitutions described in the IARC germline database for (i) transactivation capability and (ii) capacity to reduce the activity of the wild-type allele (i.e., DN effect) using a quantitative yeast-based assay. The functional classifications of p53 alleles were then related to clinical variables. We confirmed that a classification based on transactivation alone can identify familial cancer cases with more severe clinical features. Classification based on DN effects allowed us to highlight similar associations but did not reveal distinct clinical subclasses of SD alleles, except for a correlation with tumor tissue prevalence. We conclude that in carriers of germline TP53 mutations transactivation-based classification of TP53 alleles appears more important for genotype/phenotype correlations than DN effects and that haplo-insufficiency of the TP53 gene is an important factor in cancer proneness in humans. Mol Cancer Res; 9(3);

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.

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.