EFFICIENT SYSTEM OF HOMOLOGOUS RNA RECOMBINATION IN BROME MOSAIC-VIRUS - SEQUENCE AND STRUCTURE REQUIREMENTS AND ACCURACY OF CROSSOVERS

Brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants engineered to support intersegment RNA recombination, was used for the determination of sequence and structural requirements of homol ogous crossovers. A 60-nucleotide (nt) sequence, common between wild-t ype RNA2 and mutant RNA3, supported efficient repair (90%) of a modifi ed 3' noncoding region in the RNA3 segment by homologous recombination with wild-type RNA2 3' noncoding sequences. Deletions within this seq uence in RNA3 demonstrated that a nucleotide identity as short as 15 n t can support efficient homologous recombination events, while shorter (5-nt) sequence identity resulted in reduced recombination frequency (5%) within this region. Three or more mismatches within a downstream portion of the common 60-nt RNA3 sequence affected both the incidence of recombination and the distribution of crossover sites, suggesting t hat besides the length, the extent of sequence identity between two re combining BMV RNAs is ah important factor in homologous recombination. Site-directed mutagenesis of the common sequence in RNA3 did not reve al a clear correlation between the stability of predicted secondary st ructures and recombination activity. This indicates that homologous re combination does not require similar secondary structures between two recombining RNAs at the sites of crossovers. Nearly 20% of homologous recombinants were imprecise (aberrant), containing either nucleotide m ismatches, small deletions, or small insertions within the region of c rossovers. This implies that homologous RNA recombination is not as ac curate as proposed previously. Our results provide experimental eviden ce that the requirements and thus the mechanism of homologous recombin ation in BMV differ from those of previously described heteroduplex-me diated nonhomologous recombination