Biomedical subjects
Publications and source records attributed to Iu S Lazurkin.
The review considers the properties and some applications of complexes formed by peptide nucleic acid (PNA) molecules with complementary DNA sites, with emphasis on the structure and properties of PNA2/DNA triplexes.
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The degree of strand separation (mu) of shear-degraded phage T2 DNA on melting was investigated. The average size of fragments (n) varied in the range from 1000 to 300 base pairs. The obtained relationship between mu and 1/n was compared with the relationship between melting temperature depression (delta Tm) and 1/n. Both relationships are nonlinear. The correlation between the relationship delta Tm(1/n) and mu0,5(1/n) enables to believe that nonlinearity of the relationship delta Tm(1/n) is governed by the process of strand separation of DNA fragments on melting.
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A formaldehyde-produced fixation of defects caused by a despiralizing action of a protein was studied in the case of DNA-RNAase A complex. The concentration of the defects fixed was measured by kinetic formaldehyde method (KF-method). It was shown that following processes take place in the complex in the presence of formaldehyde: (a) fixation of defects; (b) unwinding of DNA; (c) inactivation of the protein. The rates of all these processes depend on the concentration of formaldehyde, phi. At formaldehyde concentrations above some critical value phic the protein is inactivated before the defects are fixed. At phi less than phic the protein inactivation proceeds more slowly than the fixation of defects; at sufficiently low formaldehyde concentration no inactivation of protein occurs practically during the fixation time (20 min). The number of new defects formed during the time of fixation is linear with the formaldehyde concentration in the region where no inactivation of the protein occurs. Therefore the initial concentration of defects can be determined through an extrapolation to zero concentration of formaldehyde. On the basis of the data obtained a method is proposed for the evaluation of the number of defects in DNA caused by the despiralizing action of proteins. A model is proposed describing the behaviour of the complexes of DNA with despiralizing proteins in the presence of formaldehyde.
It has been shown that formaldehyde has no marked physical effect upon DNA resulting in lowering of its melting temperature. The effect of lowering of DNA melting temperature observed earlier by other authors resulted from the process of unwinding of DNA due to chemical reactions of formaldehyde with reactive base groups.
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Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.
The progress in understanding the phenomenon of molecular melting (helix -- coil transition) of DNA is considered. It is shown that the theory of DNA melting has reached such a stage of development when it is capable to compute melting profiles and denaturation maps for DNA with any given nucleotide sequence, on the base of a rather simple but adequate model of DNA. The effect of fine structure of the DNA melting profiles, its origin and possible applications are considered in greater detail. It is demonstrated that a direct comparison between experimental and theoretical melting profiles for the open replicative form of the upsilon X-174 phage DNA for which the complete nucleotide sequence has been published recently, confirms the theory and opens new possibilities for further investigation of equilibrium and kinetic properties of DNA molecules.
The effect of single- and double-stranded breaks in DNA phage T2, on the melting temperature of this DNA in the 0,05 M SSC solution, was investigated. The number of cleavages per 1000 nucleotide pairs varied in the range of 0 to 10. It is shown that single- and double-stranded breaks affect the melting temperature with approximately (within 20%) the same efficiency. The relationship between the melting temperature shift (delta Tm) and the number of cleavages is non-linear. The magnitude of the effect is characterized by delta Tm of 2 +/- 0.4 degrees C for the average inter-cleavage distance of 200 base pairs. It is shown that the observed melting curves are non-equilibrium ones, which is probably due to the fact that the effect of cleavages on the melting temperature is largely results from the complete and practically irreversible separation of strands.
Interaction between DNA and limited hydrolysate of T4 gene 32 protein has been studied. This limited hydrolysate is obtained when 8000 dalton segment of native 32 protein is removed proteolitically, its molecular weight being 26,000 dalton. It is shown that the melting temperatures of DNA as well as that of the synthetic homopolymer poly[d(AT)] complexed with this modified protein are more than 60 degrees lower than that of pure DNA and poly[d(AT]. The secondary structure defects in DNA promote its unwinding by the modified 32 protein. As follows from the analysis of the melting curves of the modified 32 protein -- DNA complex, this protein cooperatively binds to denatured DNA. The binding constants and cooperativity parameters are found to be equal to 10(8)--10(10) M-1 AND 10(3) respectively. It is shown that the gene 32 protein in the same environmental conditions does not destabilized native DNA and poly[d(AT)].
Even a small amount of formaldehyde is shown to induce a drop in the RNase A enzymatic activity. This drop is rapid from the start and then begins to be slower. A supposition was made on nature of the enzyme activity. Comparison of the effects of formaldehyde on the enzymatic and the destabilizing activity of RNase A was made. The effect of formaldehyde on the enzymatic activity does not correlate with its effect on the ability of RNase to destabilize the DNA double helix.
It is shown that the kinetics of DNA despiralization in the presence of beta-alanine--formaldehyde reaction product (beta-ALA-FORM) can be described in terms of theory of DNA despiralisation by "slowly reacting agents". Conditions are determined in which beta-ALA-FORM product can be used to establish the concentration of defects in DNA secondary structure. Possible advantages are discussed of using the new agent in the kinetic method of determining DNA defects as compared to formaldehyde, in particular in analysis of DNA complexes with proteins. The data obtained throw some light on the nature of the interaction between beta-alanine and formaldehyde in slightly acidic solutions and with the excess of aminoacid.
We studied the kinetics of formaldehyde dissociation from hydroxymethylated amino groups of nitrous bases on native DNA. Compared with monomers, the rate constant of formaldehyde dissociation from such bases integrate in the double helix proved to be 20 times smaller for adenine and 4 times smaller for cytidine within the temperature range of 15 to 40 degrees C. The kinetic pattern suggests that the dissociation of formaldehyde from hydroxymethylated amino groups does not occur in the direction of the base plane nor through a full fluctuational opening of base pairs. It is presumed that formaldehyde dissociation from modified amino groups is due to softer fluctuational changes which however, make it possible for formaldehyde to attack amino groups perpendicularly to the base plane.
The rate constants of forward and reverse reactions between methylol derivatives of beta-alanine and deoxycytidine 5'-phosphate, deoxyadenosine 5'phosphate and deoxyguanosine 5'phosphate and the equilibrium constants of these reactions were determined by the spectrophotometric method at 39,5 degrees C and pH 6,95. Besides, the equilibrium constant of the reaction between beta-alanine and formaldehyde was determined. Unlike deoxycytidine and deoxyadenosine 5'-phosphates, interaction of deoxyguanosine 5'phosphate with methylol derivatives is more complicated. A model proposed for the interaction of deoxyguanosine 5'phosphate with methylol derivatives explains the behavior of this nucleotide in the reaction. The kinetic and equilibrium constants of the interaction of methylol derivatives with nucleotides investigated exceed by two or three orders of magnitude the corresponding constants of the interaction of formaldehyde with these nucleotides.
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