No abstract

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase-DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme-substrate complexes were observed.

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.