Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease

Abstract

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.

Dnmt3a-CD Is Less Susceptible to Bulky Benzo[a]pyrene Diol Epoxide-Derived DNA Lesions Than Prokaryotic DNA Methyltransferases

“…Therefore, v 1 and v 2 were converted to apparent catalytic rate constants ( k cat,1 and k cat,2 , respectively) by dividing these values by the corresponding initial E 4 S concentrations. The latter was calculated in each case from the total enzyme and substrate concentrations using the K d1 and K d2 values determined from the fluorescence titration experiments (Table 2), which was justified by the fact that the FAM label does not affect the DNA-MTase interactions (27). In experiments measuring v 1 , nearly all of added substrate was present in the form of the E 4 S complex, whereas 32–88% of the added enzyme formed E 4 S complexes in the experiments designed to determine v 2 .…”

Genome-wide H3K9 Histone Acetylation Profiles Are Altered in Benzopyrene-treated MCF7 Breast Cancer Cells

“…We chose to specifically target histone modifications in our experiments rather than DNA methylation in part because DNA adducts formed by BaP metabolites may bias the ability of microarray-based studies that rely on methylation enzymes to define differential methylation patterns. In particular, BaP metabolites preferentially form adducts at methylated CpGs and have been shown to inhibit methylation-sensitive restriction enzymes that are used to generate differential methylation displays (32).…”

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

“…Despite being comprehensive, the approach is very straightforward inasmuch as it does not rely on commonly used procedures, such as restriction enzyme digestion or PCR amplification of DNA, for detecting aberrant DNA methylation. The latter two procedures are known to be impeded by the presence of bulky adducts in lesion-bearing DNA [39], [40], [41], [42]. To further provide proof of evidence on the utility of MIRA-assisted microarray approach for characterizing DNA methylation patterns in the genome, we also verified the validity of the data obtained by our MIRA-assisted microarray analysis using the well-established COBRA [32] and bisulfite sequencing methods [33].…”

“…Additional concerns include technical uncertainties surrounding the applied DNA methylation detection systems. For instance, application of a restriction enzyme-based, PCR–dependent microarray approach for studying DNA methylation in B[ a ]PDE-treated cells has proved unsuccessful [22] due to the potential interference of B[ a ]PDE-DNA adducts with restriction enzyme digestion and/or PCR-amplification steps involved therein [39], [40], [41], [42].…”