TY ELEMENT RETROTRANSPOSITION IN SACCHAROMYCES CEREVISIA

我们的研究工作旨在了解Ty元素逆转录的机制和后果。反转录转座子是一类与逆转录病毒（如HIV-1）在结构和复制模式上相似的转座子。Ty元素亲缘关系也包括哺乳动物和植物基因组的很大一部分。由于这些元素存在于酿酒酵母（Saccharomyces cerevisiae）这一高度发达的真核生物模型系统中，因此它们是研究反转录转位许多方面的范例。今年，我们报告了我们最近在转录和翻译后水平上处理调节Ty1反转录转位的宿主基因的工作。MGA2和SPT23在酿酒酵母中是功能和基因上冗余的同源物。这两个基因都涉及到一个基因子集的转录，包括Ty反转录转座子和Ty诱导的突变。这两种基因都不是生长所必需的，但mga2 spt23双突变体是不可存活的。我们已经分离出基因特异性激活因子SWI5和酵母的δ -9脂肪酸去饱和酶OLE1，作为mga2 spt23温度敏感突变（spt23-ts）的多拷贝抑制因子。当mga2 spt23-ts突变体在37℃孵育时，不饱和脂肪酸水平下降35-40%。电镜下可见内、外核膜分离，膜间可见囊泡样突起。油酸和棕榈油酸催化产物，抑制mga2 spt23-ts和mga2 spt23致死性，恢复正常核膜形态。此外，在缺乏野生型Mga2p和Spt23p的情况下，OLE1转录物的水平降低了15倍以上。我们的研究结果表明Mga2p/Spt23p通过刺激OLE1转录来控制细胞活力。真核生物基因组含有潜在的不稳定序列，其重排威胁到基因组的结构和功能。本研究表明，核苷酸切除修复(NER)/TFIIH解旋酶基因RAD3和SSL2 （RAD25）的某些突变等位基因具有合成致死率，并通过增加短序列重组和Ty1反转录转位来破坏酿酒酵母基因组的稳定性。rad3-G595R和ssl2-rtt突变不会显著改变Ty1 RNA或蛋白水平，也不会显著改变靶点特异性。然而，这些突变导致断裂的DNA分子和未整合的Ty1 cDNA的物理稳定性增加，从而导致更高水平的短序列重组和Ty1反转录转位。我们还研究了细胞同源重组功能在Ty1反转录转位中的作用。我们发现，在RAD52重组修复途径基因突变的细胞中，转位增加，但在错配修复（MSH2）或NER (RAD1或RAD2) DNA修复功能突变的细胞中，转位增加。与NER/TFIIH突变体一样，RAD52组突变体中Ty1反转录转位的增加与Ty1 cDNA水平的显著升高相关。总之，我们的研究结果将核心NER/TFIIH复合物的组成部分与同源重组/DNA双链断裂修复所需的功能与基因组稳定性联系起来，并通过涉及DNA降解的机制对Ty1反转录转位进行宿主防御。

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.