TARGETING OF THE SITE OF NONHOMOLOGOUS GENETIC-RECOMBINATION IN BROMEMOSAIC-VIRUS

The genome of brome mosaic virus (BMV) consists of three positive stra nd RNA segments that share a high degree of sequence homology in the 3 ' noncoding region. The phenomenon of both homologous and nonhomologou s intersegment RNA-RNA recombination has been demonstrated within the 3' noncoding region of BMV RNAs. It has been postulated that nonhomolo gous crossovers occur at local heteroduplexes formed between the recom bining BMV RNA substrates of the same polarity and that the formation of double-stranded regions facilitates strand switching by the replica se. To test the hypothesis of hybridization-mediated recombination in BMV, RNA-3 constructs carrying short antisense RNA1-derived sequences have been used to induce nonhomologous recombination events between RN A-1 and RNA-3 at or near the site of hybridization. We find that both the incidence of recombination and the location of recombinant junctio ns depends on the structure and the stability of heteroduplexes. Furth ermore, our preliminary results demonstrate that mutations in the heli case-like domain of BMV protein 1a affect the location of recombinant junctions. This provides experimental evidence that BMV replicase prot ein 1a participates in recombination.