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Genetic analysis of the Rad51D gene. The key event in recombination repair of DNA double strand breaks (DSB) is the formation of the Rad51 nucleoprotein filament, which is necessary for the homology search and exchange of DNA strands. An important role in the regulation of the assembly, stabilization, and disassembly of Rad51 filaments is played by the Rad51 paralogs — proteins structurally similar to Rad51. Recently, an interest Rad51 paralogs has greatly increased due to their significant role in carcinogenesis. Mammals have six paralogs of the Rad51: Rad51B, Rad51C, Rad51D, XRCC2;, XRCC3 and the SWSAP1 (RadA homolog). The Drosophila genome contains the Rad51 ortholog spnA and four Rad51 paralogs: spnB (XRCC3 homolog), spnD (hRad51C), XRCC2 and Rad51D. However, the functions of these proteins remain largely unclear. The rad201G1 mutation (radiation sensitive 201) was isolated from a natural population by its larval hypersensitivity to ionizing radiation (Khromykh, Zakharow, 1981). Subsequently, the rad201G1 mutation was extensively characterized genetically in respect of its effects on meiotic and mitotic recombination, spontaneous and radiation-induced chromosome aberrations, mutagenesis, radiation induced effects in oogenesis and in the development. Here we show that the rad201G1 mutation is caused by the insertion of the Opus retrotransposon at the 5 ’ untranslated region of the Rad51D gene. In addition to the Opus insertion in the site of mutation, the 'rad201G1' chromosome contains a number of nucleotide changes, which cause K61E, V93A and Y108H subtitutions in the Rad51D and F50L in the protein encoded by the overlapping CG42382 gene. We isolated spontaneous reversions of the rad201 radiation sensitivity phenotype. All reversions are associated with the loss of Opus, leaving the nucleotide substitutions in Rad51D and CG42382 genes intact. In the rad201G1 mutant embryos the Rad51D transcription is 30-fold reduced by contrast with the wild type or revertants, while the level of the CG42382 transcription does not differ . Thus, the rad201G1 mutation is a Rad51D allele. By contrast with the other studied members of the Rad51 family in Drosophila, Rad51D mutant has a very weak spindle phenotype which appears only with age. The results of the studies of genetic interactions between Rad51D[rad201] and spn-A mutations will be presented. A known genetic effects of the rad201G1 mutation will be reviewed in a light of the fact that they reflect the functions of the Drosophila Rad51D gene.

Evidence is now overwhelming that partially assembled states of the nucleosome are as important as the canonical structure for the understanding of how DNA accessibility is regulated in cells. We use a combination of atomistic simulation and Atomic Force Microscopy (AFM) to propose models of key partially assembled structures of the nucleosome at atomic detail: the heaxasome ((H3·H4)2·(H2A·H2B)), the tetrasome ((H3·H4)2), and the disome (H3·H4). Despite large-scale fluctuations of the “free” DNA not in direct contact with the histones in these structures, the histone-DNA contacts are stable, establishing the basis for interpretation of the role of these structures in providing variable degree of DNA accessibility within the chromatin. We show how the atomistically detailed partially assembled nucleosome structures can be used to interpret experimental observations, both in-vitro and in-vivo. Specifically, interpretation of FRET, AFM, (and SAXS) experiments under conditions where partially assembled nucleosome states are expected is greatly facilitated by the availability of atomic- resolution structural ensembles of these states. We also suggest an alternative interpretation of a recent genome-wide study that investigated types of DNA protection in classical “active” chromatin, lending support for the transcription speed-up scenario that involves partially assembled sub-nucleosome structures.

Fluorinated beta-lactams are useful building blocks for synthesis of fluorinated nitrogen-containing compounds. In addition, they can be of interest as potential pharmaceuticals and agrochemicals. Within the framework of our research of synthetic potential of fluoroaziridines (which can be easily prepared via reaction sequence involving generation of fluorocarbene by reduction of CHFBr2 with active lead in the presence of Bu4N+Br-, addition of carbene to azomethine and 1,3-cyclisation of resulting iminiofluoromethanide) we found conditions for isomerization of fluoroaziridines to alfa-fluoroimines. Isomerisation of 2,2-diphenylaziridines proceeds smoothly in dichloromethane at 40°C in the presence of SbF3 as a catalyst, while for the isomerisation of 2-alkyl-2-phenylaziridines more harsh conditions (dichloroethane, 70°C) are required. The reaction of the resulting imines with generated in situ ketene gives the side chain fluorinated beta-lactams in 22-62% yield. Unsymmetrically substituted ketenes (phenoxy- and acetoxyketene) stereoselectively give rise to cis-beta-lactams. The reaction can be performed as one-pot procedure without isolation of the imine.

A simple and convenient route to fluorinated propargyl amines and beta-lactams was developed based on isomerisation of fluoroaziridines to alfa-fluoroimines as a key step.

Beta-Lactams bearing a Ph2CF substituent at the C(4)-atom were synthesized from N-alkyl-2-fluoro-3,3-diphenylaziridines. The transformation was realized using SbF3-mediated isomerization of the monofluoroaziridines to fluorinated aldimines, followed by Staudinger reaction with ketenes. It was shown that this reaction sequence can be performed as a one-pot procedure.