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. Interestingly, neither of these genes is required for recombination between the cDNA and homologous plasmid sequences. Although rad52 and rad1 represent two distinct epistasis groups, the rad52rad1 double mutant does not show a synergistic decrease in RNA-mediated recombination. The rad51, rad55, and rad57, mitotic recA analogs, are not required for RNA-mediated recombination. In fact, mutation of rad51 increases RNA-mediated recombination. Further, 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. Preliminary observations support the hypothesis that overexpression of TOP1 increases RNA-mediated recombination by making the chromosome a better substrate for insertion of the cellular sequences by Ty1 integrase. That is, chromosomal insertions are still observed when rad52 is mutated (required for homologous chromosomal insertion, see above) in a TOP1 overexpression strain. These chromosomal insertions are likely to be mediated by Ty1 integrase. Finally, we have shown that the yeast model system can be used as an in vivo assay for reverse transcriptase activity. A human LINE element has been used as the source of reverse transcriptase for RNA-mediated recombination. Surprisingly, expression of the LINE element reverse transcriptase under the control of a galactose-inducible promoter is sufficient for RNA-mediated recombination. This observation suggests that, in this case, viral particles are not required. Physical analysis of the cDNA should provide insight into priming and insertion by LINE elements.
