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• W2062202414 abstract "Article1 April 1998free access Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations Rainer K. Brachmann Rainer K. Brachmann Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Kexin Yu Kexin Yu Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Yolanda Eby Yolanda Eby Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Nikola P. Pavletich Nikola P. Pavletich Present address: Division of Molecular Oncology, Department of Medicine and Pathology, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Jef D. Boeke Jef D. Boeke Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Rainer K. Brachmann Rainer K. Brachmann Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Kexin Yu Kexin Yu Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Yolanda Eby Yolanda Eby Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Nikola P. Pavletich Nikola P. Pavletich Present address: Division of Molecular Oncology, Department of Medicine and Pathology, Washington University School of Medicine, St Louis, MO, 63110 USA Search for more papers by this author Jef D. Boeke Jef D. Boeke Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 Search for more papers by this author Author Information Rainer K. Brachmann2, Kexin Yu1, Yolanda Eby1, Nikola P. Pavletich3 and Jef D. Boeke1 1Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205 2Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA 3Present address: Division of Molecular Oncology, Department of Medicine and Pathology, Washington University School of Medicine, St Louis, MO, 63110 USA The EMBO Journal (1998)17:1847-1859https://doi.org/10.1093/emboj/17.7.1847 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Several lines of evidence suggest that the presence of the wild-type tumor suppressor gene p53 in human cancers correlates well with successful anti-cancer therapy. Restoration of wild-type p53 function to cancer cells that have lost it might therefore improve treatment outcomes. Using a systematic yeast genetic approach, we selected second-site suppressor mutations that can overcome the deleterious effects of common p53 cancer mutations in human cells. We identified several suppressor mutations for the V143A, G245S and R249S cancer mutations. The beneficial effects of these suppressor mutations were demonstrated using mammalian reporter gene and apoptosis assays. Further experiments showed that these suppressor mutations could override additional p53 cancer mutations. The mechanisms of such suppressor mutations can be elucidated by structural studies, ultimately leading to a framework for the discovery of small molecules able to stabilize p53 mutants. Introduction Reduction or elimination of the activity of the tumor suppressor protein p53 is a characteristic of more than half of all human cancers (Hollstein et al., 1991; Caron de Fromentel and Soussi, 1992; Harris and Hollstein, 1993; Greenblatt et al., 1994). Reduced p53 activity can result from the presence of abnormally high levels of host proteins (e.g. mdm-2), or viral proteins (e.g. high-risk human papilloma virus E6) (Vogelstein and Kinzler, 1992; Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Neil et al., 1997). However, in the majority of cancers, p53 inactivation is caused by missense mutations in one p53 allele with concomitant loss-of-heterozygosity (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Donehower and Bradley, 1993; Levine, 1997). The unusually high frequency of p53 missense mutations in human cancers can be explained by their dominant-negative effect. Interference with the remaining wild-type p53 allele leads to decreased genetic stability, loss-of-heterozygosity and thus complete abrogation of p53 function (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Hann and Lane, 1995; Brachmann et al., 1996; Ko and Prives, 1996). In addition, at least some of the same missense mutations may confer a gain-of-function phenotype (Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997). p53 is active as a homotetramer and exerts its tumor suppressor function mainly as a transcription factor by inducing, among others, genes that lead to G1 arrest or apoptosis (Donehower and Bradley, 1993; Pietenpol et al., 1994; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). Apoptosis can also be induced by p53-dependent non-transcriptional mechanisms (Caelles et al., 1994; Haupt et al., 1995; White, 1996; Hansen and Oren, 1997). Reconstitution of wild-type p53 activity to cancers should thus be of substantial therapeutic benefit, an idea that is supported by several lines of evidence. First, cell lines with functional p53 are more sensitive to commonly used anti-cancer agents (Clarke et al., 1993; Lowe et al., 1993a, b, 1994). Cancer types that are rarely associated with p53 mutations can be successfully treated even at advanced stages (Fisher, 1994; Lowe, 1995; Harris, 1996). Finally, clinical studies show that human cancers without p53 mutations are more likely to be eradicated (Harris and Hollstein, 1993; Fisher, 1994; Bergh et al., 1995; Lowe, 1995; Harris, 1996). The restoration of wild-type p53 activity to cancer cells could theoretically be achieved in two ways: one could reintroduce wild-type p53, perhaps by gene therapy (Roth et al., 1996), or one could restore wild-type function to the mutated p53 in tumors (Gibbs and Oliff, 1994; Lowe, 1995; Milner, 1995; Harris, 1996). In the second case, the presence of the mutant p53 protein could be used therapeutically in at least two ways. One approach would be to interfere with the C-terminal regulatory domain of p53 using antibodies (Halazonetis and Kandil, 1993; Hupp et al., 1993; Abarzua et al., 1995; Niewolik et al., 1995) or peptides (Hupp et al., 1995; Abarzua et al., 1996; Selivanova et al., 1997) to relieve the likely negative regulation of p53 activity. This approach could succeed with p53 mutants that retain residual activity so that their up-regulation may allow their activity to exceed the threshold required for biological effects. However, these potentially therapeutic macromolecules may be difficult to use in patients since they could conceivably activate mutant and wild-type p53 proteins indiscriminately, thus leading to unwanted side effects due to inappropriately exuberant apoptosis induced by wild-type p53 activity in normal tissues. Another approach is pharmacologically to reverse the effects of tumorigenic mutations based on the structure of the p53 core domain. The vast majority of tumor-derived p53 mutations maps to the core DNA-binding domain (core domain) and invariably these mutations result in reduction or loss of DNA binding. Based on the crystal structure of the p53 core domain–DNA complex and biochemical data (Cho et al., 1994), the tumor-derived mutations can be grouped into two classes; one maps to DNA-contacting residues and eliminates p53–DNA contacts (functional mutations), while the other, larger class affects amino acids important for the structural integrity of the DNA-binding domain. Failure of the latter class of mutants to bind DNA can be attributed to structural defects, ranging from small structural shifts to the global destabilization and unfolding of the p53 core domain (structural mutations). In principle, then, increasing the stability of the folded state of p53 or introducing additional p53–DNA contacts could restore functional activity to subsets of tumor-derived mutants. Similarly, second-site suppressor mutations that either introduce additional DNA contacts or increase the stability of the folded state of the DNA-binding domain could restore function to mutant p53 molecules. The identification of the latter type of suppressor mutations would be of particular significance, as these may be able to suppress the large structural class of tumor-derived mutations, in effect acting as global suppressors, similar to those identified with model systems such as staphylococcal nuclease (Shortle and Lin, 1985). An example of a second-site suppressor mutation that presumably introduces a new p53–DNA contact has been provided by Wieczorek et al. (1996), who relied on the crystal structure of the p53 core domain (Cho et al., 1994) to predict suppressor mutations. This strategy of rational suppressor mutation design, although promising, could generate false predictions and will almost certainly be incomplete in scope. We have taken a more systematic genetic approach in yeast to identify missense mutations able to suppress common p53 cancer mutations. This approach should yield the most potent suppressor mutations attainable. We used our previously described p53 yeast assay (Brachmann et al., 1996; Vidal et al., 1996) that assesses p53 function by its ability to bind to a p53 DNA binding site and transactivate the downstream reporter gene URA3. Using PCR mutagenesis and gap repair in yeast, we selected for several intragenic missense suppressor mutations that reverse the effects of the p53 cancer mutations V143A, G245S and R249S. The same suppressor mutations had similar effects on additional p53 cancer mutations. Remarkably, these suppressor mutations restored p53 function in mammalian cells using both reporter gene and apoptosis induction readouts. Results Identification of second-site suppressor mutations for the cancer mutations V143A, G245S and R249S The yeast assay for p53 uses a tightly regulated reporter gene, 1cUAS53::URA3, integrated into the yeast genome (Brachmann et al., 1996; Vidal et al., 1996), whose expression relies on p53 interaction with a binding site upstream of URA3. Human p53 is expressed from a yeast CEN expression plasmid under the control of a constitutive promoter (Scharer and Iggo, 1992). We used a previously described synthetic binding site that conforms to the p53 binding site consensus (el-Deiry et al., 1992). The activity of wild-type p53 is scored in two ways: URA3 expression enables the yeast reporter strain to survive on plates without uracil (Ura+), but also sensitizes yeast cells to 5–fluoroorotic acid (5-Foa). Thus, Foa sensitivity (FoaS) is the second phenotype of wild-type p53. All p53 mutants tested so far have the opposite phenotype of wild-type p53 in yeast, namely Ura−FoaR (Brachmann et al., 1996). The assay can also score an intermediate phenotype (Ura+FoaR) that reflects sufficient URA3 expression for survival on SC Ura− plates, but insufficient expression to experience the toxic effects of 5-Foa. We exploited the clear phenotypic differences between wild-type and mutant p53 in this assay to identify second-site suppressor mutations for p53 cancer mutations. The general strategy relied on PCR mutagenesis and gap repair in yeast (Figure 1). The initial design removed most of the p53 ORF from the yeast expression plasmid by restriction digestion; the resulting gap was bridged with an overlapping PCR product that contained the original cancer mutation, as well as a potential second-site mutation (Figure 1A, PCR product A). We used relatively high-fidelity standard PCR conditions and Taq polymerase, as opposed to intentionally mutagenic conditions, in order to minimize the likelihood of generating confounding multiple missense mutations. Co-transformation of both products into the yeast reporter strain led to very efficient repair of the gapped plasmid through homologous recombination with a PCR product (Muhlrad et al., 1992). The yeast transformants could then be easily assessed; those that grew on plates lacking histidine and uracil in a plasmid-dependent fashion contained an intact yeast expression plasmid (His+) with a restored p53 ORF encoding a functional p53 molecule (Ura+) (Figure 1B). Inclusion of the original cancer mutation in the PCR product resulted in a very high background of reversions of the original mutation to the wild-type amino acid (data not shown). Figure 1.Design of the PCR mutagenesis and yeast gap repair experiments. (A) In the first approach, the entire core domain of the p53 ORF was mutagenized in several yeast plasmids expressing common cancer missense mutation versions of p53 using primers JB1151 and JB1152 (PCR product A). The same plasmids were gapped with the restriction enzyme PflMI. This strategy incorporated the original mutation into the PCR product. In the second approach, regions adjacent to the p53 mutations were PCR mutagenized with the intent of reducing reversion of the original p53 mutations to the wild-type codon. For V143A, the downstream region was PCR amplified with primers JB1275 and JB1276 (PCR product B) and the expression plasmid gapped with an NcoI–StuI digestion. For G245D, G245S, R248W and R249S the upstream region was PCR amplified with primers JB1273 and JB1274 (PCR product C) and the expression plasmids gapped with BspMI and Bsu36I. (B) The PCR products and gapped plasmids were designed to have overlapping segments at both ends (ranging from 38 to 335 nucleotides in length). Co-transformation of both into the yeast reporter strain led to very efficient repair of the gapped plasmids through homologous recombination with PCR products. The yeast transformants could then be easily assessed; those that grew on plates lacking histidine and uracil in a plasmid-dependent fashion had to contain an intact yeast expression plasmid (His+) with an intact p53 ORF encoding for a functional p53 molecule (Ura+), potentially containing the original p53 missense mutation and a new second-site suppressor mutation. Download figure Download PowerPoint We therefore changed our strategy and excluded the original cancer mutation from the PCR product (Figure 1A, PCR products B or C). Because the cancer mutations were virtually assured to be present in the resultant isolates, this mutagenesis scheme was clearly superior in terms of the relative frequency of suppressor mutations. In this initial study we concentrated on the regions downstream of V143A (Figure 1A, PCR product B) or upstream of G245D, G245S, R248W and R249S (Figure 1A, PCR product C). Our experiments for G245D (12 Ura+-conferring plasmids analyzed) and R248W (four Ura+-conferring plasmids analyzed) yielded no suppressor mutations, for which there may be several explanations: (i) our screen was not sufficiently exhaustive; (ii) very few or no individual suppressor mutations for these two p53 mutants exist; or (iii) we mutagenized the wrong portion of the p53 coding region. The last possibility is supported by a recent study (Wieczorek et al., 1996). This describes a downstream suppressor mutation, T284R, obtained on the basis of modeling studies, which is able to suppress p53 mutants R248W, R273C and R273H, even though full activity in certain functional assays required further artificial activation of these mutants by removal of the very C-terminal autoregulatory domain. For V143A, G245S and R249S we analyzed 44, 88 and seven Ura+-conferring plasmids, of which two, seven and one, respectively, showed persistence of the original mutations. For V143A, the suppressor mutation N268D (two independent clones) was identified. For G245S, three suppressor mutations were isolated: T123P (four total, two independent), N239Y (two total) and S240N (one total) (Table I). For R249S, a single isolate with two missense mutations, T123A and H168R, was identified (Table I). By sequencing and subcloning fragments with putative second-site suppressor mutations, we confirmed that these mutations alone were sufficient to suppress the corresponding cancer mutations (Figure 2). Both the T123A and H168R mutations were required to suppress R249S. Neither mutation individually showed even partial suppression of R249S (Figure 2). Within the detection levels of our assay, all suppressor mutations, except T123P and S240N (phenotype Ura+FoaR instead of Ura+FoaS), led to complete restoration of wild-type p53 activity in yeast at 30°C. Figure 2.Phenotypes in yeast of intragenic second-site suppressor mutations with the cancer mutations V143A, G245S and R249S. All subcloned suppressor mutations showed the same phenotype as the initially isolated plasmids. Shown are four patches corresponding to four independent yeast transformants containing the indicated p53 expression construct. The patches were grown at 30°C on SC His−, SC Ura− and SC His− + Foa 0.1%. V143A+N268D behaved like wild-type p53 in our assay (Ura+FoaS). N239Y suppressed G245S completely (Ura+FoaS), while T123P and S240N showed only partial suppression (Ura+FoaR). T123A+H168R completely suppressed the mutant phenotype of R249S. However, neither T123A nor H168R alone showed any suppression of R249S. The controls for the upper plates were from left to right: RBy41 (reporter strain with wild-type p53), RBy57 (parent strain without p53-dependent reporter gene expressing wild-type p53), RBy287 (reporter strain with V143A) and RBy198 (reporter strain with G245S). The controls for the lower plates were from left to right: RBy41, RBy55 (reporter strain with vector control), RBy57 and RBy235 (reporter strain with R249S). Download figure Download PowerPoint Table 1. Independent intragenic suppressor mutations for the p53 cancer mutations V143A, G245S and R249S Original mutation Identification of suppressor mutations Confirmatory subcloning Mammalian activitya Suppressor amino acid changes(s) Human cancer mutation Suppressor nucleotide changes(s) Original suppressor plasmid Original suppressor strain Yeast plasmid Yeast strain Mammalian plasmid Reporter gene assays Programmed cell death assay V143A N268D No AAC→GAC pRB306 RBy284 pRB334 RBy290 pRB340 20–60 15 V143A N268D No AAC→GAC pRB307 RBy285 – – – – – (+A161A) (+GCC→GCT) G245S T123P No ACT→CCT pRB284 RBy256 pRB290 RBy262 pRB320 90–260 55 G245S T123P No ACT→CCT pRB285 RBy257 – – – – – (+M40I) (+ATG→ATA) G245S N239Y No AAC→TAC pRB282 RBy255 pRB301 RBy273 pRB325 15–120 70 G245S S240N No AGT→AAT pRB286 RBy260 pRB308 RBy286 pRB326 85–120 65 R249S T123A – ACT→GCT pRB280 RBy253 pRB298 RBy270 pRB323 40–50 45 +H168R CAC→CGC T123A No ACT→GCT – – pRB294 RBy266 – – – H168R Yes CAC→CGC – – pRB296 RBy268 – – – a Percentage of wild-type p53. All yeast plasmids are based on pRB16, the parent strain to all yeast strains is RBy33 (Brachmann et al., 1996; Vidal et al., 1996) and the mammalian plasmids are based on pCMVNeoBam (Kern et al., 1992). The phenotypes of the double and triple p53 mutants in yeast are summarized in Figures 2 and 5. The activity of the p53 mutants in mammalian assays is expressed in percentages of wild-type p53 activity. The range of percentages reflects the results with different combinations of cell lines and reporter constructs. All percentage numbers are rounded off to the nearest 5% increment. The Ura and Foa phenotypes of all strains were also tested at 25°C and 37°C since the suppressor phenotype could be temperature-dependent (data not shown). V143A+N268D at 37°C was very weakly Ura+, but still FoaS. This probably reflects a partial loss of function which is masked on Foa plates by the increased innate sensitivity of yeast to 5-Foa resulting from growth at 37°C. All suppressor mutations for G245S were Ura+FoaS at 37°C, consistent with the same explanation or an improved function of these second-site mutations at the higher temperature. G245S+T123P and G245S+S240N were inactive (Ura−FoaR) at 25°C. Modeling the basis of suppression The structure of the core domain p53 complex (Cho et al., 1994) suggested that tumorigenic mutations inactivate p53 by either altering amino acids that contact the DNA (functional mutations), or in the more frequent case, by affecting the structural integrity and stability of the DNA-binding surface or of the β-sandwich (structural mutations). The structure of the p53 core domain (Cho et al., 1994) consists of a β-sandwich that serves as a scaffold for two large loops (termed L2 and L3) and a loop–sheet–helix motif (Figure 3A). The loops and the loop–sheet–helix motif form the DNA-binding surface of p53 and provide contacts to the DNA backbone and the edges of the bases. Figure 3.Suppressor mechanisms are suggested by the structure of the wild-type p53–DNA complex. (A) Schematic representation of the wild-type p53 core domain (cyan) bound to DNA (blue) (Cho et al., 1994) highlighting in yellow the residues that are mutated in cancer that were used to select for suppressors and in red the residues where the isolated second-site suppressor mutations map. (B) The mutation of Val143 (shown in yellow) to Ala in cancer disrupts the packing of this side chain with the hydrophobic residues shown in gray and may destabilize the hydrophobic core. This mutation can be rescued by the mutation of Asn268 (colored red) to Asp. In the wild-type structure, Asn268 makes a hydrogen bond, indicated with a dotted green line, that bridges the two sheets of the β-sandwich and may contribute to the stability of the β-sandwich. Additional hydrogen bonding interactions afforded by the carboxylate on N268D is expected to further stabilize this region. (C) Gly245, whose Cα atom is shown as a yellow sphere, occurs in the L3 loop that provides one of the critical DNA contacts. This region has little space for an amino acid other than a glycine, and the G245S tumorigenic mutation thus affects the structure and stability of the L3 loop and of its DNA contact. The residues where second-site mutations suppress the G245S and several other L3 loop mutations are shown in red. In the wild-type structure, Asn239 is near the DNA, but several angstroms too far away to be making a contact; its mutation to a Tyr can readily bring this residue within contact distance to the phosphodiester backbone of the DNA. In the wild-type structure, Ser240 makes a hydrogen bond (green dotted line) with a backbone amide of the β-sandwich; its mutation to Asp may improve this hydrogen bond or allow formation of an additional one. In the wild-type structure, Thr123 is mostly solvent-exposed with no apparent structure-stabilizing role; however, the L1 loop appears to have a somewhat flexible conformation and T123A/P mutations may stabilize a conformation favorable for L1 loop–DNA contact. (D) The mutation of Arg249 (shown in yellow) to Ser would deprive the L3 loop of the hydrogen bond network (green dotted lines) that bridges the L2 and L3 loops. The mutation of the neighboring His168 on the L2 loop, shown in red, to Arg may restore components of this hydrogen bond network. Download figure Download PowerPoint In our screen for second-site suppressors, we started with scaffolding mutants as they could lead to global suppressor mutations that increase the overall stability of the folded state of the core domain. The mutants we used as a basis for isolating suppressors, V143A, G245S and R249S represent a wide range of structural defects. Val143 is involved in the packing of the hydrophobic core of the β-sandwich, and its mutation to alanine probably reduces the stability of the folded state, resulting in denaturation of the core domain (Cho et al., 1994) (Figure 3B). Gly245 and Arg249 are in the L3 loop that provides the critical Arg248–DNA contact (Cho et al., 1994). Gly245 is in a region that has little space for an amino acid other than glycine; hence its substitution by serine is predicted to affect the conformation of this loop (Cho et al., 1994; Figure 3C). The Arg249 side chain is central to a hydrogen bond network that stabilizes the L3 loop, and its mutation to serine would again be expected to affect the conformation of the L3 loop (Cho et al., 1994; Figure 3C). Both the G245S and R249S mutations, in addition to affecting indirectly the critical DNA contact made by Arg248, may also reduce the overall stability of the core domain (Cho et al., 1994). A possible basis for second-site suppression is suggested by the wild-type p53 structure. Among the suppressor mutants identified, only N268D maps to the β-sandwich, and it probably increases the stability of the β-sandwich. In the wild-type structure, Asn268 occurs on the edge of the β-sandwich, in the general vicinity but not within contact distance of Val143. Its side chain makes a backbone hydrogen bond to the other sheet of the β-sandwich (Figure 3B) and its mutation to Asp may allow it to make alternate hydrogen bonds that help keep the two sheets of the β-sandwich together. Three suppressor mutations were identified in the L3 loop and its immediate vicinity. The N239Y mutation likely introduces an additional DNA contact in the critical area where the L3 loop makes its primary DNA contacts (Cho et al., 1994). In the wild-type structure, Asn239 is solvent-exposed on the DNA binding surface of p53, but does not make any DNA contacts as it is several angstroms too far away from the DNA. Its mutation to a Tyr could position the hydroxyl group to within hydrogen bonding distance of the phosphodiester backbone of the DNA, and may thus result in an additional DNA contact. The S240N mutation probably introduces additional stabilizing interactions between the L3 loop and its scaffold, the β-sandwich. In the wild-type structure, Ser240 makes a hydrogen bond to a backbone amide group of the β-sandwich, and its mutation to an Asn may allow for an additional hydrogen bond to be made to a backbone carbonyl group of the β-sandwich (Figure 3C). In contrast to the N239Y and S240N suppressor mutations, whose proposed mechanism of action may allow them to suppress multiple tumor-derived mutations, the mechanism of action of H168R is likely to be specific for the R249S tumor-derived mutant. R249S is predicted to eliminate a guanidinium group that bridges the L2 and L3 loops via hydrogen bonds, and the H168R suppressor mutation would introduce a guanidinium group in the immediate vicinity that may restore some of these interactions (Figure 3D). This proposal is consistent with the observation that in the absence of the R249S mutation, H168R interferes with p53 activity in our assay (data not shown). Furthermore, only the H168R mutation has been found in tumors, whereas none of the other suppressor mutations, as expected, has been associated with tumors (Cariello et al., 1996; Hollstein et al., 1996). The remaining two suppressor mutations both map to Thr123 at the L1 loop of the loop–sheet–helix motif. In the wild-type structure, the L1 loop provides a single DNA contact (Lys120) although its structure appears to be significantly more flexible than the L3 loop (Cho et al., 1994). Thr123 is solvent-exposed, and it does not seem to be involved in any stabilizing interactions with other portions of p53 (Cho et al., 1994). Therefore it is not clear how its mutation to Ala or Pro could improve the DNA-binding activity of p53, although a likely mechanism is that these substitutions stabilize a loop conformation that is more favorable for the Lys120–DNA contact. It is noteworthy that T123A was previously identified as a mutation that singly increases the DNA-binding affinity of p53 (Freeman et al., 1994). Evaluation of V143A, G245S and R249S with their suppressor mutations for transcriptional activity in human cell lines To validate that second-site suppressors isolated in yeast function in human cells, all p53 cancer mutations—with and without their respective suppressor mutations—were evaluated in mammalian reporter gene assays. The transient transfection experiments were initially performed in the osteosarcoma cell line Saos-2 and the lung cancer cell line H1299, both of which are p53-null (Haffner and Oren, 1995). A luciferase reporter was used with the following upstream DNA binding sites: a tandem array of 13 p53 binding sites (PG13-Luc; Kern et al., 1992; el-Deiry et al., 1993), an array of 15 mutated p53 binding sites (MG15-Luc; Kern et al., 1992; el-Deiry et al., 1993) or the p53 response element of p21/WAF1 (WWP-Luc; el-Deiry et al., 1993). The latter downstream gene is required for p53-mediated G1 arrest. All transcriptional activity readouts were normalized by co-transfection with a lacZ expression plasmid to account for variable factors such as transfection efficiency and cell death. Using PG13-Luc as the reporter plasmid, V143A and R249S showed no transcriptional activity as expected; G245S showed ∼25% residual activity as compared with wild-type p53 (Figure 4A, PG13-Luc). Consistent with the findings in yeast at 37°C, V143A+N268D showed increased but not wild-type levels of transcriptional activity as compared with V143A. T123P and S240N clearly suppressed the effect of the G245S cancer mutation. Surprisingly, G245S+N239Y showed transcriptional activity comparable with that of G245S alone (Figure 4A, PG13-Luc). T123A+H168R showed suppression of R249S, resulting in 40% of wild-type p53 trans" @default.
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• W2062202414 date "1998-04-01" @default.
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• W2062202414 title "Genetic selection of intragenic suppressor mutations that reverse the effect of common p53 cancer mutations" @default.
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