Biochemical and structural studies of stereochemically distinct benzo[a]pyrene dioepoxide DNA adducts: Effects of the methyl group in 5-methylcytosine on the conformation and nucleotide excision repair of trans-anti-[BP]-N2-dG adducts in a CpG sequence c

Mutations in the p53 gene are among the most common genetic alterations in human cancers. A disproportionate number of mutations are found in certain codons of the p53 gene, most at CpG dinucleotide sequences, which are highly methylated in human tissues. The reactivities of the metabolite of benzo[ a]pyrene, r7,t8-dihydroxy-t9,10-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to form guanine adducts at the exocyclic amino group (trans-anti-BPDE- N2-dG, or G*) are enhanced when the cytosine in CpG* sequences in DNA is methylated at its 5-position (5MeCpG*). This study was performed in order to determine if methylation might also affect the physical and/or conformational characteristics, and the nucleotide excision repair efficiencies of trans-anti-BPDE-N 2-dG adducts in site-specifically modified double-stranded oligo-2 '-deoxyribonucleotides. Spectroscopic evidence is presented that suggests that the (+)-trans-anti-BPDE-N 2-dG assumes an intercalated conformation in the 5MeCpG*, although it has a minor groove conformation in the CpG* sequence context. While such a conformational switch is not observed in the case of the stereoisomeric (+)-trans adduct that is generally positioned in the minor groove of DNA, fluorescence quenching experiments suggest that the BPDE residue is less solvent-exposed in the 5MeCpG* than in CpG* sequences.; Nucleotide excision repair (NER) assays with the prokaryotic UvrABC system indicate that incision activities are enhanced by factors of typically 2--3 by methylation of the cytosines flanking both (+)-trans- and (-)-trans-anti-BPDE-N2 -dG adducts on the 5'-side positioned within codon 273 of the human p53 gene. The enhancements are consistently greater in the case of the (-)-trans than the (+)- trans adduct. This difference is attributed in part to a conformational change from minor groove to an intercalated conformation in the case of the (-)-trans adduct upon methylation. However, the smaller though enhanced incision rates in the case of the (+)-trans adduct suggest that the methyl group in 5MeCpG* sequence context by itself enhances incision rates that are associated with a still unknown mechanism. Methylation also enhances the excision of the (+)-trans adduct positioned in the same codon 273 p53 sequence context by a factor of ∼2 in NER experiments conducted with eukaryotic human cell extracts from HeLa cells in vitro.