ИСТИНА

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), is a carcinogen and a common environmental pollutant. It is metabolized to highly genotoxic diol epoxides (B[a]PDE) that react chemically with guanine. 6-thioguanine (SG) is a widely used antileukemia agent. We investigated whether B[a]PDE-dG or SG at CpG sites in DNA perturbed cytosine-methylation by the de novo methyltransferase, Dnmt3a. Duplex 30- mer oligos containing stereoisomeric B[a]PDE-dG adducts or SG residues were prepared and incubated with the catalytic domain of murine Dnmt3a (Dnmt3a-CD). Methylation rates were significantly reduced by trans-B[a]PDE-dG (minor groove adducts), regardless of their position in the substrate, and by cis-B[a]PDE-dG (intercalated adducts) within the CpG site. In Dnmt3a-CD/B[a]PDE-DNA complexes, stereochemistrydependent fluorescence enhancement of the covalently bound B[a]PDE residues was observed. We suggest that the movement of the Dnmt3a-CD catalytic loop, and flipping of the target cytosine, are disturbed by the bulky B[a]PDE residues when they are in the minor groove. The impact of intercalated adducts arises from the distortion of the CpG site. Methylation also decreased when SG replaced guanine within the CpG site. This effect might be due to disturbance of the Dnmt3a-CD contact to the 6-oxo group of the guanine, a contact predicted to be important for the formation of the catalytically competent Dnmt3a-CD/DNA complex (1). The SG absorption band is above 300 nm where DNA and the enzyme are transparent, allowing us to follow methylationassociated conformational changes in SG-DNA by circular dichroism. The results suggested disturbances of base stacking at CpG sites within Dnmt3a-CD/SG-DNA complexes. Other forms of damaged guanine such as 8-oxoguanine and O6- methylguanine were also found to have significant effects on the Dnmt3a-catalysed methylation (2,3). The effect of this damage diminishes as follows: 8-oxoG >> (+)-trans-B[a]PDE-dG ~ (+)-cis-B[a]PDE-dG > O6meG > SG. Our data suggest that damage to guanine can affect epigenetic processes that depend upon Dnmt3a-methylation.