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Effects of Benzo[a]pyrene−Deoxyguanosine Lesions on DNA Methylation Catalyzed by EcoRII DNA Methyltransferase and on DNA Cleavage Effected by EcoRII Restriction Endonuclease†
journal contribution
posted on 2005-01-25, 00:00 authored by Vladimir B. Baskunov, Fedor V. Subach, Alexandr Kolbanovskiy, Marina Kolbanovskiy, Sergei A. Eremin, Francis Johnson, Radha Bonala, Nicholas E. Geacintov, Elizaveta S. GromovaDNA methylation is an important cellular mechanism for controlling gene expression. Whereas
the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons,
have been widely studied, little is known about their influence on DNA methylation. We have constructed
site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts
derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (−)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts,
either (+)- or (−)-trans-anti-B[a]P−N2-dG (G*), positioned either at the 5‘-side or the 3‘-side
deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5‘-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on
complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII
restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl
group to the C5 position of the 3‘-side cytosine of each strand of the recognition sequence, whereas
R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes
and the catalytic cleavage were completely abolished when G* was positioned at the 3‘-side dG position
(5‘-...CCTGG*...). When G* was at the 5‘-side dG position, binding was moderately diminished, but
cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5−30 but
the catalytic activity was either abolished or reduced 4−80-fold when the adducts were located at either
position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes.
These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in
chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression
of genes that promote tumor development.