Publications by authors named "O P Lukashevich"

Background: Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase.

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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.

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Benzo[a]pyrene (B[a]P) is a well-characterized environmental polycyclic aromatic hydrocarbon pollutant. In living organisms, B[a]P is metabolized to the genotoxic anti-benzo[a]pyrene diol epoxide that reacts with cellular DNA to form stereoisomeric anti-B[a]PDE-N(2)-dG adducts. In this study, we explored the effects of adduct stereochemistry and position in double-stranded DNA substrates on the functional characteristics of the catalytic domain of murine de novo DNA methyltransferase Dnmt3a (Dnmt3a-CD).

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To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane.

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In this article we report on construction of expression vector, heterologous expression in Escherichia coli, isolation, purification, and physicochemical characterization of an artificial chimeric protein HMWb(5)-EGFP consisting of full-length cytochrome b(5) (HMWb(5)) and green fluorescence protein (EGFP) from Aequorea. Optimization of expression conditions yielded an expression level up to 1500 nmol of chimeric protein per liter of culture. Recombinant chimeric protein HMWb(5)-EGFP was purified from cell membranes by using metal-affinity chromatography.

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