title: Impact of 7,8-dihydro-8-oxoguanine on methylation of the CpG site by Dnmt3a.
abstract: 7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.
authors.affiliation: Chemistry Department and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia.
authors.forename: Diana V
authors.initials: DV
authors.lastname: Maltseva
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.forename: Elizaveta S
authors.initials: ES
authors.lastname: Gromova
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018899:CpG Islands / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects*; D000090205:DNA Methyltransferase 3A; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D013997:Time Factors
chemical_list: C453560:7,8-dihydro-8-oxoguanine; C000715535:Dnmt3a protein, mouse; D006147:Guanine; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D019793:Fluorescein