Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.     

Characteristics of two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two genomic RNA copies is essential for the assembly of retrovirus particles. This process has been studied in detail, and a two-step mechanism has been proposed for the human immunodeficiency virus type 1 (HIV-1). A similar model can be assumed for avian sarcoma and leukosis viruses (ASLV), despite the lack of homology between the dimerization initiation site (DIS) of ASLV and that of HIV-1. The structural features of the ASLV DIS were studied with the examples of the avian leukosis virus HPRS-103 and the avian sarcoma virus CT-10. The rate of spontaneous transition from loose to tight dimers at a higher temperature was studied as dependent on the stem length in the DIS hairpin. Dimers of both types were formed by the selected RNA fragments of the two viruses. The conditions of loose dimer formation differed considerably, although the two viruses had identical sequences (5-A-CUGCAG-3) of the hairpin loop. Dimerization of CT-10 RNA fragments required an RNA concentration at least an order of magnitude higher than in the case of HPRS-103. The difference was explained by deletion of an adenine from the hairpin stem of C-10.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 147–154.Original Russian Text Copyright © 2005 by Beniaminov, Samokhin, Ulyanov, Minyat.     

Conformational dynamics in mixed alpha/beta-oligonucleotides containing polarity reversals: a molecular dynamics study using time-averaged restraints

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3-3 and 5-5 phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two -containing duplexes – d(GCGAAT-3-3-T-5-5-CGC)₂ and d(ATGG-3-3-T-5-5-GCTC)r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (d _av, J _rms, and J _av) and improvements in sixth-root R-factors (R ^x). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R ^x values. In both systems, the backbone torsion angles comprising the 3-3 linkage as well as the (high S-) sugars of the -nucleotide and preceding residue (–1) are relatively static, while the conformations of the 5-5 linkage and the sugar in the neighboring -nucleotide (+1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.