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The methylation of cytosines at CpG sites in DNA, carried out de novo by DNA methyltransferase Dnmt3a, is a basic epigenetic modification involved in gene regulation and genome stability. Aberrant CpG methylation in gene promoters leads to oncogenesis. In oncogene promoters, CpG sites often colocalize with guanine-rich sequences capable of folding into G-quadruplexes (G4s). Our in vitro study aimed to investigate how parallel G4s formed by a sequence derived from the c-MYC oncogene promoter region affect the activity of the Dnmt3a catalytic domain (Dnmt3a-CD). For this purpose, we designed synthetic oligonucleotide constructs: a c-MYC G4-forming oligonucleotide and linear double-stranded DNA containing an embedded stable extrahelical c-MYC G4. The topology and thermal stability of G4 structures in these DNA models were analyzed using physicochemical techniques. We showed that Dnmt3a-CD specifically binds to an oligonucleotide containing c-MYC G4, resulting in inhibition of its methylation activity. c-MYC G4 formation in a double-stranded context significantly reduces Dnmt3a-CD-induced methylation of a CpG site located in close proximity to the quadruplex structure; this effect depends on the distance between the non-canonical structure and the specific CpG site. One would expect DNA hypomethylation near the G4 structure, while regions distant from this non-canonical form would maintain a regular pattern of high methylation levels. We hypothesize that the G4 structure sequesters the Dnmt3a-CD and impedes its proper binding to B-DNA, resulting in hypomethylation and activation of c-MYC transcription. [ABSTRACT FROM AUTHOR]

The impact of stereoisomeric (+)- and (-)-trans-anti-B[a]P.[N.sup.2]-dG adducts ([B.sup.+] and [B.sup.-]) on DNA methylation by prokaryotic DNA methyltransferases M.SssI and M.HhaI are investigated. The trans-anti-B[a]P-[N.sup.2]-dG lesion when it flanks a target dC in the CpG dinucleotide sequence on its 5'-side has a greater adverse impact on methylation than the same lesion when it is 3' adjacent or opposite to the target dC.

Research has been conducted on the nonregular heteropyrimidine sequence oligonucleotides that can form self-associated parallel-stranded structures. The identification of these structures via neutral and acidic pH UV melting transitions and other methods has been carried out.

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis. [ABSTRACT FROM AUTHOR]

A study is conducted to assess the effects of 7,8-dihydro-8-oxoguanine (8-oxoG) on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). Based on the results it is concluded that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, to favor a nonproductive binding mode.

The ways in which methylation is impacted by a pair of stereoisomeric polycyclic aromatic hydrocarbon carcinogen-modified DNA adducts positioned at either one or the other of the guanine sites in EcoRII recognition sequence are reported. The results 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 might allow the expression of genes that promoter tumor development.

The impact of bulky carcinogen- DNA adducts positioned at or near recognition sites ( Cp 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- 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 Cp G site] and when it is intercalated on the 5′-side of the Cp G 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 Cp 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• Dnmt3a- CD complexes. In the presence of the regulatory factor Dnmt3 L, an enhancement of both methylation rates and fluorescence was observed, which is consistent with a Dnmt3 L-mediated displacement of the catalytic loop towards the Cp G site. [ABSTRACT FROM AUTHOR]

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[ a]pyrene (B[ a]P), (+)-7 R,8 S-diol 9 S,10 R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)- trans-B[ a]P- N2-dG and, to a lesser extent, (+)- cis-B[ a]P- N2-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+) -cis-B[ a]P- N2-dG and (+)- trans-B[ a]P- N2-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[ a]P residues are positioned in different microenvironments in the DNA–protein complexes. We have previously shown that the (+)- trans-isomeric adduct inhibits both the binding and methylating efficiencies ( kcat) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry 45, 6142–6159]. Here we show that the stereoisomeric (+)- cis-B[ a]P- N2-dG lesion has only a minimal effect on the binding of these MTases and on kcat. The minor-groove (+)- trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)- cis adduct does not interfere with the minor-groove DNA–catalytic loop contacts, allowing near-normal binding of the MTases and undiminished kcat values. [ABSTRACT FROM AUTHOR]

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII. [ABSTRACT FROM AUTHOR]

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis., Competing Interests: Authors declare no conflict of interest in financial or any other sphere.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.