Role of RNA structure in non-homologous recombination between genomic molecules of brome mosaic virus

Brome mosaic virus (BMV) is a tripartite genome, positive-sense RNA virus o f plants. Previously it was demonstrated that local hybridization between B MV RNAs (RNA-RNA heteroduplex formation) efficiently promotes non-homologou s RNA recombination. In addition, studies on the role of the BMV polymerase in RNA recombination suggested that the location of non-homologous crossov ers depends mostly on RNA structure. As a result, a detailed analysis of a large number of non-homologous recombinants generated in the BMV-based syst em was undertaken. Recombination hot-spots as well as putative elements in RNA structure enhancing non-homologous crossovers and targeting them in a s ite-specific manner were identified. To verify these observations the recom binationally active sequence in BMV RNA3 derivative was modified. The resul ts obtained with new RNA3 mutants suggest that the primary and secondary st ructure of the sequences involved in a heteroduplex formation rather than t he length of heteroduplex plays the most important role in the recombinatio n process. The presented data indicate that the sequences proximal to the h eteroduplex may also affect template switching by BMV replicase, Moreover, it was shown that both short homologous sequences and a hairpin structure h ave to accompany a double-stranded region to target non-homologous crossove rs in a site-specific manner.