DNA mobility in crystals of a nonspecific lambda cro/DNA complex.

DNA location in the crystal of the nonspecific lambda cro/(GT)4.(AC)4 complex has been studied by the isomorphous replacement method using iodinated and brominated oligonucleotides. The results of the search for heavy atom positions combined with previously obtained molecular replacement data suggest that the DNA octamer occupies two overlapping positions, each of the two duplexes (GTGTGTGT).(ACACACAC) belonging to the same imaginary longer double helix and differing only in the shift by two base pairs along the common sugar-phosphate backbone. In the crystals of the heavy atom derivatives different orientations of the DNA octamer are observed as well. It seems reasonable that the DNA mobility of both kinds might be a common feature of crystals of nonspecific repressor/DNA complexes.

A simplified multidimensional search used for crystal structure solution of pCpGpCpGpCpG with two duplexes pre asymmetric unit.

A simplified multidimensional search was applied to determine the structure of deoxyhexamer CGCGCG in the crystal form belonging to the space group C222(1) (a = 45.6 A, b = 37.3 A, c = 70.3 A). This crystal form contains two Z-DNA duplexes per asymmetric unit in a similar orientation. The search consists of several main steps. As a first step, the analysis of packing modes is carried out and constraints for the position of duplexes in asymmetric unit are formulated. In order to choose true packing mode duplexes are represented as cylinders of a constant density and global search is carried out. Only reflections belonging to the plane in reciprocal space perpendicular to DNA axis were used in the calculations. An analytical representation of diffraction from a hollow cylinder is given which allows further refinement of corresponding parameters. During the global search the value of a linear correlation coefficient is estimated for each solution to pick up the best one. At the second step the "more local" search is carried out. The DNA model of a certain conformation (in our case Z-DNA) is taken and several parameters are being varied. These are: a) rotations of the independent duplexes around their axes (only two rotational parameters for two duplexes in our example) and b) a few positional parameters for which there is no corresponding constraint in the packing mode tested. In our case we had to vary only x1 and y2, where subindex corresponds to molecule number. The last stage concerned is quite similar to the multidimensional search realised in program ULTIMA. The only difference that the number of variable parameters is less in our search. This number for the case under consideration was 4 instead of 12 that would be necessary in a conventional multidimensional search.

[New packing of B-DNA in crystals]. / Novaia upakovka B-DNK v kristallakh.

Two crystal forms of the self-complementary tetramer GpGpCpC have been obtained by phase diagram technique: P6(2)22/P6(4)22. a = b = 67.7 A, c = 105.6 A and P3(2)12/P3(1)12, a = b = 116.9 A. c = 116.4 A. Both crystals form diffract at least up to 3.2 A. Diffraction patterns of both crystal forms have strongest base-stacking reflections corresponding to the Bragg spacing 3.38 A which is typical for B-DNA. Moreover the self-rotation function of the first crystal form shows regular located two-fold pseudo-axes periodicity of which also indicates that this is B-conformation. The same conclusion can be reached on the basis of the crystal packing of the duplexes in the unit cell. It should be emphasized that this is a new example of B-DNA crystal packing.

[Use of phase diagrams in crystallization of oligonucleotide duplexes. II. Setting of the crystallized samples]. / Ispol'zovanie fazovykh diagramm pri kristallizatsii oligonukleotidnykhdupleksov. II. Postanovka kristallizatsionnykh prob.

Oligonucleotide crystallization technique based on the method of phase diagrams is described in detail with (pGpT)3.(pApC)3 hexamer as an example. The key point of the technique consists of dividing the multiparameter crystallization space into a set of regions, each of which corresponds to the precipitation of a duplex in complex with a certain number of counterions.

Phase diagrams for DNA crystallization systems.

Phase diagrams for several oligonucleotide duplex-spermine systems have been constructed. These diagrams characterize the duplex and spermine concentrations ranges in which crystalline precipitates are formed. All of them are wedge-like form. The slope of the upper branch of the diagram is determined by the oligonucleotide length. The position of the lower branch depends on both the nucleotide sequence and its length. The position of the lower branch depends on both the nucleotide sequence and its length. It has been shown that the addition to the system of MgCl2 and NaCl salts and MPD results in specific changes in the diagrams. A model for oligonucleotide duplex-spermine system has been suggested which explains the main characteristic features of the obtained phase diagrams. The experimental phase diagrams for the (pGpT)n (pApC)n-spermine system (n = 2,3,4) have been analyzed ion terms of this model and the values of the binding constants of spermine and Mg2+ ions binding to duplexes have been determined. It permitted to identify the complexes that precipitated in different regions of the phase diagrams under various conditions. The diagram obtained in the presence of a cobalt hexammine counterion is also considered. It has been shown that this phase diagram, in general, is similar to those obtained for the oligonucleotide duplex-spermine system.

[The length of DNA determines the degree of regularity of crystals of the cro-repressor complex]. / Dlina DNK opredeliaet stepen' uporiadochennosti kristallov kompleksa s cro-repressorom.

The DNA-cro-repressor complex crystals have been obtained, five DNA fragments of the same nucleotide sequence and different length being used. The rotation function for crystals of complexes with hexamer (pGpT)3 . (pApC)3 and with octamer (pGpT)3 . (pApC)3 have been calculated. The order of cro-DNA complex crystals is shown to vary with DNA length, the crystal of the complex with octamer being the most perfect among all investigated complexes.

[The use of phase diagrams in the crystallization of oligonucleotide duplexes. I. A model of crystallization of the (pGpT)n.(pApC)n + spermine system]. / Ispol'zovanie fazovykh diagramm pri kristallizatsii oligonukleotidnykh dupleksov. I. Modelirovanie povedeniia kristallizatsionnoi sistemy "(pGpT)n.(pApC)n + spermin".

A set of experimental phase diagrams revealing the region of existence of microcrystals in mixture "(pGpT)n.(pApC)n+spermine", n = 2,3,4, was obtained. All diagrams are wedge-like with the slope of the upper branch and the level of the lower one depending on the oligonucleotidd length. The presence of MPD, MgCl2 and NaCl changes the form of the diagrams in a different manner. A model explaining the peculiar features of the diagrams for mixture "oligonucleotide duplex+spermine" is proposed. The analysis of the diagrams was carried out on the basis of this model and the values of the binding constants for binding of spermine and Mg2+ to duplexes were estimated. Some conclusions about the types of complexes, which may form microcrystals in different regions of diagrams were made.

[Effect of formaldehyde on the enzymatic activity of RNAase A]. / Vliianie formal'degida na fermentativnuiu aktivnost' RNKazy A.

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.

[Influence of formaldehyde on the melting temperature of DNA]. / Vliianie formal'degida na temperaturu plavleniia DNK.

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.