Impact of Benzo[a]pyrene-2‘-deoxyguanosine Lesions On Methylation Of DNA by SssI and HhaI DNA Methyltransferases

Abstract

DNA damage caused by the binding of the tumorigen 7R,8S-diol 9S,10R-epoxide (B[a]PDE), a metabolite of bezo[a]pyrene, to guanine in CpG dinucleotide sequences could affect DNA methylation and, thus, represent a potential epigenetic mechanism of chemical carcinogenesis. In this work, we investigated the impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P-N(2)-dG adducts (B(+) and B(-)) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI. These two methyltransferases recognize CpG and GCGC sequences, respectively, and transfer a methyl group to the C5 atom of cytosine (C). A series of 18-mer unmethylated or hemimethylated oligodeoxynucleotide duplexes containing trans-anti-B[a]P-N(2)-dG adducts was generated. The B(+) or B(-) residues were introduced either 5' or 3' adjacent or opposite to the target 2'-deoxycytidines. The B[a]PDE lesions practically produced no effect on M.SssI binding to DNA but reduced M.HhaI binding by 1-2 orders of magnitude. In most cases, the benzo[a]pyrenyl residues decreased the methylation efficiency of hemimethylated and unmethylated DNA by M.SssI and M.HhaI. An absence of the methylation of hemimethylated duplexes was observed when either the (+)- or the (-)-trans-anti-B[a]P-N(2)-dG adduct was positioned 5' to the target dC. The effects observed may be related to the minor groove conformation of the bulky benzo[a]pyrenyl residue and to a perturbation of the normal contacts of the methyltransferase catalytic loop with the B[a]PDE-modified DNA. Our results indicate that a trans-anti-B[a]P-N(2)-dG lesion flanking a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.

Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene‐modified DNA by fluorescence methods

“…The impact of (+)- and (-)- trans -B[ a ]PDE- N 2 -dG adducts and (+)- cis -B[ a ]PDE- N 2 -dG adduct on methylation of 18-mer DNA duplexes by M.SssI was studied earlier [7, 8]. However, in the present work, we focused on the impact of two types of intercalative adducts that were not examined before.…”

“…The DNA substrates contained overlapping recognition sites of M.HhaI (5′-GCGC), M.SssI (5′-CG) and Dnmt3a (5′-CG). It was shown that the MTase binding affinities for 18-mer canonical DNA and 18-mer (+)- and (-)- trans -B[ a ]PDE-DNA decrease in the order M.HhaI > M.SssI > Dnmt3a-CD, with K d values in the range 10 pM – 70 nM [7, 8, 14]. The enhancement of fluorescence intensities (I 384 ) by a factor 45-50 relative to protein-free B[ a ]PDE-DNA duplexes was observed in the case of G C G t+ C/C GM G•М.SssI•AdoHcy, G t+ C G C/C GM G•M.HhaI•AdoHcy, and G C G t+ C/C GM G•M.HhaI•AdoHcy complexes with (+)- trans -B[ a ]PDE -N 2 -dG adducts positioned within the MTase recognition sites (Fig.…”

“…In contrast, B[ a ]PDE-modified nucleotides that flank the CpG sequences on their 5′-side is crucial for the prokaryotic CpG-recognizing MTase M.SssI. In this case, methylation was either abolished in 18-mer duplexes containing B[ a ]PDE residues in the minor groove (motif A) [7], or strongly decreased in the case of 30-mer duplexes with the base-displaced intercalative (-)- cis -B[ a ]PDE- N 2 -dG adduct (motif B) (Table 3). The B[ a ]PDE- N 6 -dA adducts in the same position have less impact on the methylation rates of M.SssI.…”

“…These studies have shown that (1) the B[ a ]PDE lesions affect primarily the methylation reaction by decreasing the efficiency of the catalytic step rather than the binding of the enzymes to the carcinogen-DNA adducts, and that (2) the methylation efficiency is strongly dependent on adduct conformation and its position within the DNA substrate. For prokaryotic M.SssI, the most significant effect on the methylation efficiency was observed when DNA containing (+)- and (-)- trans -B[ a ]PDE- N 2 -dG adducts, that are known to be positioned in the minor groove [9], were positioned on the 5′-side of the CpG recognition site of the enzyme [7]. However, a number of important questions remain that have not been resolved.…”

Dimeric bisbenzimidazoles inhibit the DNA methylation catalyzed by the murine Dnmt3a catalytic domain

“…In addition, disposition of Hoechst 33258 in the DNA minor groove was demonstrated for type I and II complexes with poly(dGdC)·poly(dG-dC) 28 . Recently we found that the presence of a bulky benzo[a]pyrene residue in the DNA minor groove caused a dramatic decrease in prokaryotic MTases SssI and HhaI methylating capacity because of distortion of DNA contacts with the enzyme catalytic loop 29,30 . Superimposition of the DNA methyltransferase HhaI (M.HhaI-DNA) complex structure onto the Dnmt3a-CD structure showed high similarity of M.HhaI and Dnmt3a-CD catalytic loop conformations 7 .…”