We are using a yeast model system to study the reverse transcription of cellular mRNAs and the subsequent recombination event involving the cDNA, a process referred to as RNA-mediated recombination. Because a cellular RNA is reverse transcribed, the reverse transcriptase must be provided in trans, for example, by a retrovirus (LTR-containing) or LINE element (nonLTR- or polyA-containing retrotransposon). We have shown that the LTR-containing, yeast retrotransposon, Ty1 can provide the source of reverse transcriptase. Ty1 is also required for priming reverse transcription of the cellular mRNA and priming occurs by a template switch. This has led to the following model: the cellular transcript is packaged into the viral particle along with the Ty1 transcript. Ty1 initiates reverse transcription on its own genome, using its normal priming mechanism, and then a template switch occurs onto the poly(A) tail of the cellular mRNA. Reverse transcription extends to the end of the transcript and a second template switch occurs back to Ty1 sequences, in this case directed by short regions (2-9 base pairs) of complementarity. The cellular sequences are thereby embedded in Ty1 sequences, thus, providing an alternative mechanism for oncogene capture by retroviruses. The cDNA can be inserted by Ty integrase or by the host recombination machinery. We have found that RAD52 and RAD1 (cellular genes representing two distinct recombination and repair pathways in yeast) are both required for recombination between the cDNA and homologous chromosomal sequences. The requirement for RAD1 is believed to reflect a role for RAD1 in removing nonhomologies from the 3' end of the cDNA. Interestingly, neither of these genes is required for recombination between the cDNA and homologous plasmid sequences, indicating that plasmid and chromosomal RNA-mediated gene conversion events utilize distinct subsets of cellular genes. RAD51, RAD55, and RAD57, mitotic RecA analogs, are not required for RNA-mediated recombination. In fact, mutation of RAD51 increases RNA-mediated recombination. We have shown that overexpression of the yeast topoisomerase I gene increases RNA-mediated recombination 100-fold. Expression of Ty1 is slightly increased, but Ty1 transposition is greatly increased in a TOP1 overexpression strain. The overexpression of TOP1 might increase RNA-mediated recombination by making the chromosome a better substrate for insertion of the cellular sequences by Ty1 integrase. In support of this hypothesis is the finding that chromosomal insertions are still observed when rad52 is mutated (required for homologous chromosomal insertion, see above) in a TOP1 overexpression strain. In addition, chromosomal insertions are reduced when Ty1 integrase is mutated. Finally, we have observed interference between Ty1 and Ty3 (distinct families of yeast LTR retrotransposons). This interference is observed even if Ty3 is competent only for particle formation. The mechanism of interference is being further investigated.
