Molecular construction (superstructures) with adjustable properties based on double-stranded nucleic acids

Formation of molecular construction that consists of double-stranded molecules of nucleic acids (or synthetic polynucleotides) located at the distance 30-50 A in the spatial structure of particles of their cholesteric liquidcrystalline dispersion and crosslinked by polymeric chelate bridges is described. The resulting superstructure, which possesses peculiar physicochemical properties, could be used as integral biosensor whose properties depend on temperature, the presence of chemical or biologically active compounds of different nature, etc.     

Distribution functions, describing the binding of extended ligands with DNA molecules. Possible use for cases of DNA condensation

Due to noncooperative binding of ligands to DNA molecules, DNA molecules are in equilibrium with different numbers of adsorbed ligands. This equilibrium for a given concentration of the free ligand in the solution is characterized by the distribution function, which describes the probability of revealing the DNA molecule with a definite number of adsorbed ligands. If polycations act as ligands, DNA molecules with the number of ligands sufficient for neutralizing the charges on phosphates may undergo a phase transition. One example of this transition is the formation of liquid-crystalline dispersions during the binding of DNA to chitosan. We analyzed the binding of chitosan to DNA on the assumption that this binding is due to equilibrium adsorption. At a definite concentration of chitosan in solution, DNA molecules are in equilibrium with different numbers of adsorbed molecules of chitosan. If the number of adsorbed ligands exceeds some critical value, the DNA molecule covered with chitosan becomes capable of interacting with other DNA molecules. As a result of this interaction (attraction), liquid-crystalline dispersions can form. Equations describing the dependence of the concentration of DNA molecules on the concentration of the ligand in solution were derived. It was shown that, at given parameters of the model, it is possible to describe experimental data characterizing the formation of cholesteric liquid-crystalline dispersions. The analysis of the data makes it possible to reconstitute both the size of the binding site occupied by chitosan on the DNA and the energy of interaction of chitosan with DNA.     

M.BstF5I-4, the Forth DNA-Methyltransferase of the BstF5I Restriction–Modification System from Bacillus stearothermophilus F5

The fourth DNA-methyltransferase of the BstF5I restriction–modification (RM) system from Bacillus stearothermophilus F5 (M.BstF5I-4) was discovered, which modifies the adenine residue within the upper strand of the recognition site 5"-GGATG-3"/5"-CATCC-3". Thus, unlike other known RM systems, the BstF5I RM system comprises four genes encoding DNA-methyltransferases, three of which possess the same substrate specificity and methylate adenine within the 5"-GGATG sequence.     

M.BstF5I-4, the forth DNA methyltransferase of BstF5I restriction-modification system from Bacillus stearothermophilus F5

The fourth DNA-methyltransferase of the BstF5I restriction-modification (RM) system from Bacillus stearothermophilus F5 (M.BstF5I-4) was discovered, which modifies the adenine residue within the upper strand of the recognition site 5'-GGATG-3'/5'-CATCC-3'. Thus, unlike other known RM systems, the BstF5I RM system comprises four genes encoding DNA-methyltransferases, three of which possess the same substrate specificity and methylate adenine within the 5'-GGATG sequence. The English version of the paper.     

The Kinetic Mechanism of Phage T4 DNA-[N6-Adenine]-Methyltransferase

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the GATC recognition site catalyzed by the phage T4 DNA-[N6-adenine]-methyltransferase (MTase) [EC 2.1.1.72] showed that the reverse reaction is at least 500 times slower than the direct one. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAMDNAmetDNASAH (S-adenosyl-L-homocysteine). Pronounced inhibition was observed at high concentrations of the 20-meric substrate duplex, which may be attributed to formation of a dead-end complex MTase–SAH–DNA. In contrast, high SAM concentrations proportionally accelerated the reaction. Thus, the reaction may include a stage whereby the binding of SAM and the release of SAH are united into one concerted event. Computer fitting of alternative kinetic schemes to the aggregate of experimental data revealed that the most plausible mechanism involves isomerization of the enzyme.     

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.     

Principles of creating biotransducers based on nucleic acid liquid crystals

A brief concept of biosensors is presented. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating sensing units for biosensors using different principles of molecular recognition are considered. General schemes of sensing units based on liquid-crystalline dispersions formed from low-molecular mass DNA, its complexes with "cross-linking" agents and from circular superhelical DNA molecules are described.     

The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague

The LcrV protein (V-antigen) is a multifunctional virulence factor in Yersinia pestis, the causative agent of plague. LcrV regulates the translocation of cytotoxic effector proteins from the bacterium into the cytosol of mammalian cells via a type III secretion system, possesses antihost activities of its own, and is also an active and passive mediator of resistance to disease. Although a crystal structure of this protein has been actively sought for better understanding of its role in pathogenesis, the wild-type LcrV was found to be recalcitrant to crystallization. We employed a surface entropy reduction mutagenesis strategy to obtain crystals of LcrV that diffract to 2.2 A and determined its structure. The refined model reveals a dumbbell-like molecule with a novel fold that includes an unexpected coiled-coil motif, and provides a detailed three-dimensional roadmap for exploring structure-function relationships in this essential virulence determinant.     

Symmetry elements in DNA structure important for recognition/methylation by DNA [amino]-methyltransferases

The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in k_cat was observed for all three enzymes if a particular element of structural symmetry was disrupted. For the BamHI MTase, integrity of the ATCC was critical, while an intact GAT sequence was necessary for the activity of T4Dam, and an intact GA was necessary for EcoDam. Theoretical alignment of the region of best contacts between the protein and DNA showed that in the case of a palindromic interaction site, a zone covering the 5′-symmetric residues is located in the major groove versus a zone of contact covering the 3′-symmetric residues in the minor groove. Our data fit a simple rule of thumb that the most important contacts are aligned around the methylation target base: if the target base is in the 5′ half of the palindrome, the interaction between the enzyme and the DNA occurs mainly in the major groove; if it is in the 3′ half, the interaction occurs mainly in the minor groove.     

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.