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

Using a yeast based p53 functional assay we previous:ly demonstrated that t he WC-induced p53 mutation spectrum appears to be indistinguishable from th e one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (c odon 248, CpG site) represented the major hot spot in NMSC but was not foun d mutated in the yeast system. In order to determine whether UVC-induced mu tagenic events may be facilitated at methylated cytosine (5mC), a yeast exp ression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII e ndonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradi ated (lambda(max): 254nm) and transfected into a yeast strain containing th e ADE2 gene regulated by a p53-responsive promoter. The results revealed th at: (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 meth ylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-in duced p53 mutation spectra derived from methylated and unmethylated pLS76 w ere indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to e stimate their relatedness (P = 0.85); (iv) the presence of 5mC did not incr ease 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 cau se 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 hut may have a "sponta neous" origin. (C) 1999 Elsevier Science B.V. All rights reserved.