5-Methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation

Paola Monti, Alberto Inga, Gina Scott, Anna Aprile, Paola Campomenosi, Paola Menichini, Laura Ottaggio, Silvia Viaggi, Angelo Abbondandolo, Philip A. Burns, Gilberto Fronza

Research output: Contribution to journalArticle

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Abstract

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (λ(max): 254nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P=0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solar light and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a 'spontaneous' origin. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)93-103
Number of pages11
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume431
Issue number1
DOIs
Publication statusPublished - Dec 16 1999

Fingerprint

5-Methylcytosine
Skin Neoplasms
Melanoma
Yeasts
Mutation
Codon
Deoxyribonuclease HpaII
Cytosine
Mutagenesis
Methylation
Complementary DNA
Light
Survival
Genes

Keywords

  • 5-methylcytosine
  • Molecular epidemiology
  • p53
  • Ultraviolet light

ASJC Scopus subject areas

  • Molecular Biology
  • Health, Toxicology and Mutagenesis

Cite this

5-Methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation. / Monti, Paola; Inga, Alberto; Scott, Gina; Aprile, Anna; Campomenosi, Paola; Menichini, Paola; Ottaggio, Laura; Viaggi, Silvia; Abbondandolo, Angelo; Burns, Philip A.; Fronza, Gilberto.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 431, No. 1, 16.12.1999, p. 93-103.

Research output: Contribution to journalArticle

Monti, P, Inga, A, Scott, G, Aprile, A, Campomenosi, P, Menichini, P, Ottaggio, L, Viaggi, S, Abbondandolo, A, Burns, PA & Fronza, G 1999, '5-Methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation', Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, vol. 431, no. 1, pp. 93-103. https://doi.org/10.1016/S0027-5107(99)00187-6
Monti, Paola ; Inga, Alberto ; Scott, Gina ; Aprile, Anna ; Campomenosi, Paola ; Menichini, Paola ; Ottaggio, Laura ; Viaggi, Silvia ; Abbondandolo, Angelo ; Burns, Philip A. ; Fronza, Gilberto. / 5-Methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation. In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis. 1999 ; Vol. 431, No. 1. pp. 93-103.
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abstract = "Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98{\%} protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (λ(max): 254nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P=0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solar light and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a 'spontaneous' origin. Copyright (C) 1999 Elsevier Science B.V.",
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AU - Monti, Paola

AU - Inga, Alberto

AU - Scott, Gina

AU - Aprile, Anna

AU - Campomenosi, Paola

AU - Menichini, Paola

AU - Ottaggio, Laura

AU - Viaggi, Silvia

AU - Abbondandolo, Angelo

AU - Burns, Philip A.

AU - Fronza, Gilberto

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N2 - Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (λ(max): 254nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P=0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solar light and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a 'spontaneous' origin. Copyright (C) 1999 Elsevier Science B.V.

AB - Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (λ(max): 254nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P=0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solar light and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a 'spontaneous' origin. Copyright (C) 1999 Elsevier Science B.V.

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