The roles of transcription and genotoxins underlying p53 mutagenesis in vivo

Abstract

Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.

Fig. 1.

(A) An example of mfg computer output. The C nucleotide (red) at mfg nt 233 was selected and the most stable SLS (ΔG, −5.0) in which that base is unpaired is shown. The output also provides the window size, percent unpaired, ΔG and mutability index. (B) Transcription of the p53 gene, resulting in negative supercoiling in the wake of the transcription complex. The SLS formed (with a ΔG of −5.0) contains hypermutable codon 282. (C) Correlation between mutation frequencies of the twelve most hypermutable bases in p53 (39) and the mfg-predicted percent unpaired for each base, using a window size of 43 nts. The hypermutable base of codon 282 is arrowed. Excel was used to calculate R² values, which are statistically significant when P ≤ 0.05. (D) Activation of the p53 promoter as measured by Chloramphenicol AcetylTransferase reporter activity as benzo[a] pyrene (B[a]P) concentration increases (4). (E) Induction of the p53 promoter as measured by fold increase in luciferase reporter activity with increasing concentrations of the genotoxin daunomycin (2). (F) Increase in p53 messenger RNA over time as a function of increased exposure time to genotoxin mitomycin C (5). For (C–F) n values are 13, 4, 4 and 3, respectively.

Fig. 2.

(A and B) Correlation between rates of transcription and mutation in Escherichia coli auxotrophs leuB (30) and argH (31). (C and D) Correlation between mutation rates and transcription in a Green Fluorescent Protein transgene in the human pre-B cell line 18–81, due to the activation of transcription by doxycycline (dox) (27) and in the VHB1-8 antibody gene (29). Linear regression values were calculated in Excel. R² values are statistically significant when P ≤ 0.05. For (A–D) n values are 10, 4, 4 and 3, respectively.

Fig. 3.

Examination of the fate of mutable Gs in p53 in all cancers compared with lung cancer. (A) The relative frequencies of p53 G-to-T mutations by tissue type. (B) The fate of G mutations in all cancers in which a majority of mutations (74.0%) are G-to-A (intrinsic). (C) The fate of G mutations in lung cancers in which a majority (57.7%) are oxyradical-induced G-to-T transversions. Mutation data are derived from missense mutations in the eight most mutable codons in the IARC TP53 database. Data for B and C are taken from Table II. (D) In all (pooled) cancers, the relative rate of G-to-A intrinsic mutations occurring in a specific G base at low levels of transcription is depicted by a 33 nt SLS and (E) in lung cancers at high levels of transcription (depicted by a 45 nt structure containing the same hypermutable G) with the approximate relative rates of G-to-A intrinsic mutations (rate of ∼1) and genotoxins-oxidized G-to-T transversions (rate of ∼3).

Fig. 4.

S1 cleavage analysis of exon 7 of p53. Plasmid DNA containing the non-transcribed strand of exon 7 and hypermutable codons 245 and 248 was analyzed by S1 endonuclease. (A) Selected mfg computer output (15°C, 44 nt window). Column 1 shows mutation frequencies of the hypermutable bases; column 2 identifies the hypermutable codon; column 3, the mfg assigned nt number; column 4, the color-coded stability (ΔG) of the predicted highest stability SLSs containing the mutable unpaired base; column 5 contains red arrows at S1 cleavage sites; column 6 shows the sequence of the non-transcribed strand; column 7 shows the complementary transcribed strand. (B) A segment of the p53 sequence identifying the relative locations of predicted color-coded SLSs in relation to the locations of cleavage sites (red font and arrows) and the hypermutable codons (black bracket). (C) Three mfg-predicted secondary structures containing codons 245 and 248 showing S1 cleavage sites in loops (red arrows). Hypermutable bases are circled (red). (D) Polyacrylamide gel showing S1 cleavage sites (red arrowed) next to the transcribed strand sequence ladder.