The reverse transcription of cellular RNAs and the subsequent insertion of the cDNA into the chromosome, a process referred to as RNA-mediated recombination, has probably played an important role in shaping the mammalian genome. The homologous or nonhomologous insertion of the reverse transcribed cellular transcript (cDNA) can provide a template for the evolution of new gene function, permit the development of alternative regulatory strategies and lead to chromosomal rearrangements. The purpose of this work is to understand the mechanism of RNA-mediated recombination and the viral and cellular functions required, using a yeast model system. We have shown that both the LTR-containing retrotransposon Ty and a LINE element can serve as sources of reverse transcriptase activity. These elements use very different priming mechanisms, and the mechanism of priming and insertion by LINE elements is not clearly understood. One hypothesis is that priming initiates from a nick in the chromosomal DNA. We have constructed a strain in which the topoisomerase I gene is overexpressed, to increase chromosomal nicking, as a tool for understanding priming and insertion of LINEs. A second aspect of the project is aimed at identifying the cellular genes involved in chromosomal insertion of the cDNA. We have developed an assay that specifically detects recombination between a diffusible cDNA and chromosomal allele. Using this assay, we have shown that RNA-mediated gene conversion is dependent on RAD52 (involved in most recombination) and RAD1 (involved in excision repair). Insertion of the cDNA can also be mediated by viral functions. We can detect reverse transcription and cDNA insertion of chromosomal transcripts using the retrotransposon Ty3. Ty3 inserts site-specifically upstream of tRNA genes. Therefore, Ty3 may provide a vehicle for gene delivery, eliminating concern for non-specific integration into the open reading frame of essential genes.
