Our research efforts are directed toward understanding the mechanism and consequences of Ty element retrotransposition. Retrotransposons are a class of transposable elements that resemble retroviruses, such as HIV-1, in their structure and mode of replication. Ty element relatives also comprise a significant fraction of mammalian and plant genomes. Ty elements are a paradigm for studying many aspects of retrotransposition because these elements are found in Saccharomyces cerevisiae, a highly developed eukaryotic model system. This year, we report on our recent work addressing host genes that modulate Ty1 retrotransposition at the transcriptional and posttranslational levels. MGA2 and SPT23 are functionally and genetically redundant homologs in Saccharomyces cerevisiae. Both genes are implicated in the transcription of a subset of genes, including Ty retrotransposons and Ty-induced mutations. Neither gene is essential for growth, but mga2 spt23 double mutants are inviable. We have isolated a gene-specific activator, SWI5, and the delta-9 fatty acid desaturase of yeast, OLE1, as multicopy suppressors of an mga2 spt23 temperature-sensitive mutation (spt23-ts). The level of unsaturated fatty acids decreases 35-40% when the mga2 spt23-ts mutant is incubated at 37o. Electron microscopy of these cells reveals a separation of inner and outer nuclear membranes that is sometimes accompanied by vesicle-like projections in the intermembrane space. The products of Ole1p catalysis, oleic acid and palmitoleic acid, suppress mga2 spt23-ts and mga2 spt23 lethality, and restore normal nuclear membrane morphology. Furthermore, the level of the OLE1 transcript decreases more than 15-fold in the absence of wild-type Mga2p and Spt23p. Our results suggest that Mga2p/Spt23p control cell viability by stimulating OLE1 transcription. Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality, and destabilize the Saccharomyces cerevisiae genome by increasing both short sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels, or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short sequence recombination and Ty1 retrotransposition. We have also examined the role of the cellular homologous recombination functions on Ty1 retrotransposition. We find that transposition increases in cells mutated for genes in the RAD52 recombinational repair pathway, but not in cells mutated in DNA repair functions dedicated to mismatch repair (MSH2) or NER (RAD1 or RAD2). Like the NER/TFIIH mutants, the increase in Ty1 retrotransposition in mutants of the RAD52 group is correlated with a marked increase in the level of Ty1 cDNA. Together, our results link components of the core NER/TFIIH complex and functions required for homologous recombination/DNA double-strand break repair with genome stability, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.
