Abstract
Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.
Highlights
Regardless of the stereochemistry of anti-BPDE, it is highly reactive, and the major adducts are formed by the cis or trans opening of the epoxide at the C-10 position by the exocyclic amine of guanine [10]
It is clear that methylation of the CpG sequence leads to enhanced BPDE reactivity with the difference ranging from 3- to 4-fold over the range of BPDE concentration
Effect of Methylation on the 3Ј-Side of the BPDE Adduct or in the Complementary Strand—To determine if the specific sequence or position of the 5-mC relative to the modified guanine was important in inducing the presumed structural change that was measured in the native Polyacrylamide gel electrophoresis (PAGE) analysis (Fig. 5), an oligonucleotide was prepared in which there was a cytosine on both the 5Ј- and 3Ј-side of the guanine (Fig. 6A) along with versions of this sequence where either the 5Ј- or 3Ј-C was methylated
Summary
B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene diol epoxide; (Ϯ)-anti-BPDE, ((Ϯ)-anti-r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-dGuo, benzo[a]pyrene adduct at the N-2 position of guanine; N-AAAF, N-acetoxy-N-acetyl-2aminofluorene; 5-mC, 5-methylcytosine; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis. It is well established that this gene is mutated in nearly one-half of all cancers [24] and contains six codons that are major mutagenic hot spots Five out of these six are in methylated CpG sequences [25], and when the DNA binding sites for BPDE were measured in the p53 sequence in human lung cells [26] or in plasmid DNA [22] it was found that binding was targeted to these same sequences and that methylation of the 5Ј-C was required for the enhanced reactivity [22]. It was found that the specific presence of both a 5-mC and a (ϩ)-trans-B[a]P-dGuo adduct (the major adduct formed in vivo) resulted in a substantial change in the DNA Structure and that the level of this adduct was increased when the CpG was methylated
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