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Probing murine methyltransfease D nmt3a interactions with benzo[ a ]pyrene‐modified DNA by fluorescence methods
Author(s) -
Minero Antonio S.,
Lukashevich Olga V.,
Cherepanova Natalia A.,
Kolbanovskiy Alexander,
Geacintov Nicholas E.,
Gromova Elizaveta S.
Publication year - 2012
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2012.08756.x
Subject(s) - benzo(a)pyrene , chemistry , pyrene , food science , organic chemistry
The impact of bulky carcinogen‐ DNA adducts positioned at or near recognition sites ( C p G ) of eukaryotic DNA methyltransferases on their catalytic activities is poorly understood. In the present study, we employed site‐specifically modified 30‐mer oligodeoxyribonucleotides containing stereoisomeric benzo[ a ]pyrene diol epoxide ( B [ a ] PDE )‐derived guanine ( B [ a ] PDE ‐ N 2 ‐ dG ) or adenine ( B [ a ] PDE ‐ N 6 ‐ dA ) adducts of different conformations as substrates of the catalytic domain of murine D nmt3a ( D nmt3a‐ CD ). The fluorescence of these lesions was used to examine interactions between D nmt3a‐ CD and DNA . In B [ a ] PDE ‐ DNA • D nmt3a‐ 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 ‐ N 2 ‐ dG adducts in the C p G site] and when it is intercalated on the 5′‐side of the C p G site [(+)‐ trans ‐ B [ a ] PDE ‐ N 6 ‐ dA adduct]. The fluorescence of B [ a ] PDE ‐modified DNA • D nmt3a‐ CD complexes exhibits only small changes when the B [ a ] PDE is intercalated with base displacement in (+)‐ and (−)‐ cis ‐ B [ a ] PDE ‐ N 2 ‐ dG adducts and without base displacement in the (−)‐ trans ‐ B [ a ] PDE ‐ N 6 ‐ dA adduct. The initial rates of methylation were significantly reduced by the minor groove trans ‐ B [ a ] PDE ‐ N 2 ‐ dG adducts, regardless of their position in the substrate and by the intercalated cis ‐ B [ a ] PDE ‐ N 2 ‐ dG adducts within the C p G 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 • D nmt3a‐ CD complexes. In the presence of the regulatory factor D nmt3 L , an enhancement of both methylation rates and fluorescence was observed, which is consistent with a D nmt3 L ‐mediated displacement of the catalytic loop towards the C p G site.