Benzo[a]pyrene (B[a]P) is a well-characterized environmental polycyclic aromatic hydrocarbon pollutant. In living organisms, B[a]P is metabolized to the genotoxic anti-benzo [a]pyrene diol epoxide that reacts with cellular DNA to form stereoisomeric anti-B [a]PDE-N 2 -dG adducts. In this study, we explored the effects of adduct stereochemistry and position in double-stranded DNA substrates on the functional characteristics of the catalytic domain of murine de novo DNA methyltransferase Dnmt3a (Dnmt3a-CD). A number of 18-mer duplexes containing sitespecifically incorporated (+)-and (−)-trans-anti-B[a]PDE-N 2 -dG lesions located 3′-and 5′-adjacent to and opposite the target cytosine residue were prepared. The Dnmt3a-CD binds cooperatively to the DNA duplexes with an up to 5-fold greater affinity as compared to the undamaged DNA duplexes. Methylation assays showed a 1.7-6.3 fold decrease of the methylation reaction rates for the damaged duplexes. B[a]PDE modifications stimulated a non-productive binding and markedly favoured substrate inhibition of Dnmt3a-CD independently of DNA methylation status. The latter effect was sensitive to the position and stereochemistry of the B[a]PDE-N 2 -dG adducts. The overall effect of trans-anti-B [a]PDE-N 2 -dG adducts on Dnmt3a-CD was less detrimental than in the case of the prokaryotic methyltransferases we previously investigated.Benzo [a]pyrene (B[a]P) is an ubiquitous and harmful pollutant that is abundant in car exhaust and tobacco smoke (1), and is metabolically activated to the biologically active benzo [a]pyrene-7,8-diol-9,10-epoxides with predominant formation of benzo [a]pyrene-7R, 8S-dihydrodiol-9S,10R-epoxide ((+)-anti-B[a]PDE). In the anti-orientation of B[a]PDE, the 7-OH and 9,10-epoxide groups are on opposite sites of the planar polycyclic aromatic ring system (2,3). Both the (+)-and (−)-anti-enantiomers of B[a]PDE bind covalently to the exocyclic amino group of guanine, with trans or cis opening of the epoxide ring. The most abundant adduct is (+)-trans-anti-B[a]PDE-N 2 -dG, both in vivo (4) and in vitro (about 90%) (5), with lower amounts of the (+)-cis-and (−)-trans-anti-B [a] The following buffers (B1-B7) were used: B1, 50 mM sodium phosphate (pH 6.0), 1 M NaCl, 10 mM mercaptoethanol, 10% (v/v) glycerol, 0.1% Triton X-100; B2, buffer B1 containing 17 μg/ml phenylmethanesulfonyl chloride, 5 μg/ml leupeptin and 1 μg/ml pepstatin A; B3, B4, and B5, buffer B2 containing 10 mM, 20 mM and 150 mM imidazoleHCl, respectively; B6, 20 mM Tris-HCl (pH 7.4), 0.2 mM EDTA, 2 mM dithiothreitol, 5% (v/v) glycerol; B7, 20 mM HEPES-NaOH (pH 7.0), 100 mM KCl, 1 mM EDTA, 0.2 mM dithiothreitol. Enzyme Expression and PurificationThe N-terminal His 6 tag fusion catalytic domain of Dnmt3a was expressed in E. coli BL21 (DE3) cells (Novagen) using the pET28a plasmid containing Dnmt3a-CD as a vector, as described previously (28). Cells were grown in LB medium at 32°C with intensive aeration until A 600 ~ 0.7 was attained. Protein expression was initiated by the addition of 1 mM i...

BackgroundDnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.ResultsMutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.ConclusionsWe conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

Summary The impact of bulky carcinogen-DNA adducts positioned at or near recognition sites (CpG) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In this work we have employed site-specifically modified 30-mer oligodeoxyribonucleotides containing stereoisomeric benzo[a]pyrene diol epoxide (B[a]PDE)-derived guanine (B[a]PDE-N2-dG) or adenine (B[a]PDE-N6-dA) adducts of different conformations as substrates of catalytic domain of murine Dnmt3a (Dnmt3a-CD). The fluorescence of these lesions was used to examine interactions between Dnmt3a-CD and DNA. In B[a]PDE-DNA•Dnmt3a-CD complexes the intensity of fluorescence of the covalently bound B[a]PDE residues is enhanced relative to the protein-free value when the B[a]PDE is positioned in the minor groove ((+)- and (−)-trans-B[a]PDE-N2-dG adducts in the CpG site) and when it is intercalated on the 5′-side of the CpG site ((+)-trans- B[a]PDE-N6-dA adduct). The fluorescence of B[a]PDE-modified DNA•Dnmt3a-CD complexes exhibits only small changes when the B[a]PDE is intercalated with base displacement in (+)- and (−)-cis- B[a]PDE-N2-dG adducts and without base displacement in the (−)-trans- B[a]PDE-N6-dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans-B[a]PDE-N2-dG adducts regardless of their position in the substrate and by the intercalated cis-B[a]PDE-N2-dG adducts within the CpG site. The observed changes in fluorescence and methylation rates are consistent with the flipping of the target cytosine and a catalytic loop motion within the DNA•Dnmt3a-CD complexes. In the presence of the regulatory factor Dnmt3L, an enhancement of the methylation rates and fluorescence were observed which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.

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