MUTATIONS IN THE HELICASE-LIKE DOMAIN OF PROTEIN 1A ALTER THE SITES OF RNA-RNA RECOMBINATION IN BROME MOSAIC-VIRUS

A system that uses engineered heteroduplexes to efficiently direct in vivo crossovers between brome mosaic virus (BMV) RNA1 and RNA3 (P. Nag y and J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993) has been used to explore the possible involvement of BMV 1a protein, an es sential RNA replication factor, in RNA recombination. Relative to wild -type 1a, several viable amino acid insertion mutations in the helicas e-like domain of BMV 1a protein affected the nature and distribution o f crossover sites in RNA3-RNA1 recombinants. At 24 degrees C, mutants PK19 and PK21 each increased the percentage of asymmetric crossovers, in which the RNA1 and RNA3 sites joined by recombination were not dire ctly opposite each other on the engineered RNA3-RNA1 heteroduplex used to target recombination but rather were separated by 4 to 85 nucleoti des. PK21 and another la mutant, PK14, also showed increases in the fr action of recombinants containing nontemplated U residues at the recom bination junction. At 33 degrees C, the highest temperature that permi tted infections with PK19, which is temperature sensitive for RNA repl ication, the mean location of RNA1-RNA3 crossovers in recombinants rec overed from PK19 infections was shifted by nearly 25 bp into the energ etically less stable side of the RNA1-RNA3 heteroduplex. Thus, mutatio ns in the putative helicase domain of the la protein can influence BMV RNA recombination. The results are discussed in relation to models fo r recombination by template switching during pausing of RNA replicatio n at a heteroduplexed region in the template.