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 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

Fingerprint

Benzo(a)pyrene
Epoxy Compounds
Fluorescence
DNA
Methylation
DNA Adducts
Oligodeoxyribonucleotides
Cytosine
Guanine
Methyltransferases
Adenine
Substrates
Carcinogens
Conformations
Catalyst activity
Catalytic Domain

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Minero, A. S., Lukashevich, O. V., Cherepanova, N. A., Kolbanovskiy, A., Geacintov, N., & Gromova, E. S. (2012). Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods. FEBS Journal, 279(20), 3965-3980. https://doi.org/10.1111/j.1742-4658.2012.08756.x

Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods. / Minero, Antonio S.; Lukashevich, Olga V.; Cherepanova, Natalia A.; Kolbanovskiy, Alexander; Geacintov, Nicholas; Gromova, Elizaveta S.

In: FEBS Journal, Vol. 279, No. 20, 10.2012, p. 3965-3980.

Research output: Contribution to journalArticle

Minero, Antonio S. ; Lukashevich, Olga V. ; Cherepanova, Natalia A. ; Kolbanovskiy, Alexander ; Geacintov, Nicholas ; Gromova, Elizaveta S. / Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods. In: FEBS Journal. 2012 ; Vol. 279, No. 20. pp. 3965-3980.
@article{ec4117a89ade4023b325e9c12198a4d8,
title = "Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods",
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.",
keywords = "benzo[a]pyrene-DNA, fluorescence, intercalation, methylation, minor groove",
author = "Minero, {Antonio S.} and Lukashevich, {Olga V.} and Cherepanova, {Natalia A.} and Alexander Kolbanovskiy and Nicholas Geacintov and Gromova, {Elizaveta S.}",
year = "2012",
month = "10",
doi = "10.1111/j.1742-4658.2012.08756.x",
language = "English (US)",
volume = "279",
pages = "3965--3980",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "20",

}

TY - JOUR

T1 - Probing murine methyltransfease Dnmt3a interactions with benzo[a]pyrene-modified DNA by fluorescence methods

AU - Minero, Antonio S.

AU - Lukashevich, Olga V.

AU - Cherepanova, Natalia A.

AU - Kolbanovskiy, Alexander

AU - Geacintov, Nicholas

AU - Gromova, Elizaveta S.

PY - 2012/10

Y1 - 2012/10

N2 - 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.

AB - 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.

KW - benzo[a]pyrene-DNA

KW - fluorescence

KW - intercalation

KW - methylation

KW - minor groove

UR - http://www.scopus.com/inward/record.url?scp=84867096917&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84867096917&partnerID=8YFLogxK

U2 - 10.1111/j.1742-4658.2012.08756.x

DO - 10.1111/j.1742-4658.2012.08756.x

M3 - Article

C2 - 22913541

AN - SCOPUS:84867096917

VL - 279

SP - 3965

EP - 3980

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 20

ER -