Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods.
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 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 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-N(2)-dG adducts in the CpG site] and when it is intercalated on the 5'-side of the CpG site [(+)-trans-B[a]PDE-N(6)-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-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 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 both methylation rates and fluorescence was observed, which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.
Department of Chemistry, Moscow State University, Moscow, Russia.
Antonio S
AS
Minero
Olga V
OV
Lukashevich
Natalia A
NA
Cherepanova
Alexander
A
Kolbanovskiy
Nicholas E
NE
Geacintov
Elizaveta S
ES
Gromova
D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000737:chemistry*; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
D018736:DNA Adducts; C000715535:Dnmt3a protein, mouse; D009838:Oligodeoxyribonucleotides; D001564:Benzo(a)pyrene; D006147:Guanine; D003596:Cytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000225:Adenine