Genetic analysis of the Rad51D gene

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.