Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods

Antonio S. Minero, Olga V. Lukashevich, Natalia A. Cherepanova, Alexander Kolbanovskiy, Nicholas E. Geacintov, Elizaveta S. Gromova

Research output: Contribution to journalArticle

Abstract

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-N2-dG) or adenine (B[a]PDE-N6-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-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 both methylation rates and fluorescence was observed, which is consistent with a Dnmt3L-mediated displacement of the catalytic loop towards the CpG site.

Original languageEnglish (US)
Pages (from-to)3965-3980
Number of pages16
JournalFEBS Journal
Volume279
Issue number20
DOIs
StatePublished - Oct 2012

Keywords

  • benzo[a]pyrene-DNA
  • fluorescence
  • intercalation
  • methylation
  • minor groove

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods'. Together they form a unique fingerprint.

  • Cite this