Elizaveta S. Gromova

Impact of 7,8-dihydro-8-oxoguanine on methylation of the CpG site by Dnmt3a.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.

Homology Modeling of the CG-specific DNA Methyltransferase SssI and its Complexes with DNA and AdoHcy

Abstract Prokaryotic DNA methyltransferase M. SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed “FRankensteins monster” approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.

Prokaryotic DNA Methyltransferases: The Structure and the Mechanism of Interaction with DNA

The review considers current views on the function of DNA methyltransferases (MTases) that belong to prokaryotic type II restriction–modification systems. A commonly accepted classification of MTases is described along with their primary and tertiary structures and molecular mechanisms of their specific interaction with DNA (including methylation). MTase inhibitors are also considered. Special emphasis is placed on the flipping of the target heterocyclic base out of the double helix and on the methods employed in its analysis. Base flipping is a fundamentally new type of DNA conformational changes and is also of importance in the case of other DNA-operating enzymes. MTases show unique sequence homology, and are similar in structure of functional centers and in the mechanism of methylation. These data contribute to the understanding of the general biological significance of methylation, since prokaryotic and eukaryotic MTases are structurally and functionally similar.

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non‐specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N‐terminal His residues was shown to be cross‐linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non‐cognate DNAs in the absence of cofactor, Mg2+ ions.

EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event.

EcoRII is a typical restriction enzyme that cleaves DNA using a two‐site mechanism. EcoRII endonuclease is unable to cleave DNA which contains a small number of EcoRII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of EcoRII sites. To investigate the mechanism of activation, the kinetics of stimulated EcoRII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one EcoRII recognition site (trans‐activation) by a competitive mechanism: the activator increased substrate binding but not catalysis. The activation increased if the substrate concentration decreased and if the activator had a lower affinity for the enzyme than the substrate. The introduction of the second recognition site into the 71 bp duplex also enabled cleavage of this substrate (cis‐activation). Pyrophosphate bonds were incorporated into one of two recognition sites to switch off the cleavage of the phosphodiester bonds. Analysis of cleavage products of these modified substrates showed that EcoRII cuts one of two coordinated recognition sites in one catalytic event.

Dimeric bisbenzimidazoles inhibit the DNA methylation catalyzed by the murine Dnmt3a catalytic domain

When located in the DNA minor groove, dimeric bisbenzimidazoles DB(n) effectively inhibited in vitro the Dnmt3a catalytic domain (IC50 5–77 μM). The lowest IC50 value was observed for compound DB(11) with an 11-unit methylene linker joining the bisbenzimidazole fragments. Increased time of incubation of DNA with DB(n) as well as the presence of AT-clusters in DNA enhances the inhibitory effect.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments: EcoRII endonuclease cleavage of substrates with repeated natural and modified recognition sites

Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT‐pair in the recognition site for a non‐complementary TT‐ or AA‐pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5′‐terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.

The Ecl18kI restriction-modification system: cloning, expression, properties of the purified enzymes

Ecl18kI is a type II restriction‐modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5′….↓CCNGG….′ sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow. The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity. The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer. The interactions of M.Ecl18kI and R.Ecl18kI with 1,2‐dideoxy‐d‐ribofuranose containing DNA duplexes were investigated. The target base flipping‐out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non‐canonical hydrolysis of the modified strand.

Optical rotatory dispersion and circular dichroism of mono- and oligonucleotide-amino acids (amidates)

Abstract 1. 1. Optical rotatory dispersion (ORD) and circular dichroism (CD) of 23 mono- and oligonucleotide-amino acids (amidates) have been studied. 2. 2. The ORD and CD of purine nucleotides with an aromatic amino acid or amine display the Cotton effect of the opposite sign and with a greater amplitude as compared with those of unsubstituted nucleotides. A conclusion is made about the possible non-covalent interactions between the purine base and the benzene ring of the 5′-substituent. 3. 3. ORD and CD of alanine derivatives of purine nucleotides and phenylalanine derivatives of P-Urd and P-dThd are similar to those of the respective nucleotides. In the case of the phenylalanine derivative of P-Cyd and anisidide of P-Urd, the Cotton effect has somewhat different amplitude as compared with that of P-Cyd and P-Urd. 4. 4. The Cotton effect exhibited by dThd-P-Ado-amidates has a lesser amplitude as the amine radical bound with the internucleotide phosphorus grows in size. This is accounted for by introduction of the amine radical between the planes of thymine and adenine resulting in unstacking of bases in dThd-P-Ado. 5. 5. The study of ORD and CD of uridilyl-(5′→3′)-guanilyl-(5′→N)-phenylalanine has shown that the phenylalanine residue in the 5′-terminal phosphate group is responsible for the disordering of the base stacking in this compound.

The stereochemistry of benzo[a]pyrene-2′-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)‐7R,8S‐diol 9S,10R‐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) Biochemistry45, 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.