TY ELEMENT RETROTRANSPOSITION IN SACCHAROMYCES CEREVISIA

我们的研究工作是针对了解泰元素反转录转座的机制和后果。 逆转录转座子是一类在结构和复制模式上类似于逆转录病毒（如HIV-1）的转座因子。 Ty元件亲属也包括哺乳动物和植物基因组的重要部分。 Ty元件是研究反转录转座的许多方面的范例，因为这些元件存在于高度发达的真核模型系统酿酒酵母中。 今年，我们报告我们最近的工作，解决宿主基因，调节Ty1逆转录转座在转录和翻译后水平。MGA 2和SPT 23在酿酒酵母中是功能和遗传上冗余的同源物。 这两个基因都涉及一个基因子集的转录，包括Ty逆转录转座子和Ty-induced突变。 这两个基因都不是生长所必需的，但mga2 spt23双突变体是不可活的。 我们已经分离出一种基因特异性激活剂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）的多拷贝抑制剂。 在37度下培养时，不饱和脂肪酸的水平降低了35 - 40%。 这些细胞的电子显微镜显示核膜内外分离，有时伴随着膜间隙中的囊泡样突起。 Ole1p催化的产物油酸和棕榈油酸抑制了mga2 spt23-ts和mga2 spt23的致死性，并恢复了正常的核膜形态。 此外，在不存在野生型Mga2p和Spt23p的情况下，OLE1转录物的水平降低超过15倍。 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。真核生物基因组包含潜在的不稳定序列，其重排威胁到基因组的结构和功能。 在这里，我们表明，某些突变体等位基因的核苷酸切除修复（NER）/TFIIH解旋酶基因RAD 3和SSL 2（RAD 25）赋予合成致死性，并通过增加短序列重组和Ty1逆转录转座不稳定的酿酒酵母基因组。 rad3-G595R和ssl2-rtt突变不显著改变Ty1 RNA或蛋白水平或靶位点特异性。 然而，这些突变导致断裂的DNA分子和未掺入的Ty1 cDNA的物理稳定性增加，这导致更高水平的短序列重组和Ty1逆转录转座。 我们还研究了Ty1反转录转座的细胞同源重组功能的作用。 我们发现，在RAD52重组修复途径中基因突变的细胞中，转座增加，但在专门用于错配修复（MSH2）或NER（RAD1或RAD2）的DNA修复功能突变的细胞中，转座增加。 与NER/TFIIH突变体一样，RAD 52组突变体中Ty1逆转录转座的增加与Ty1 cDNA水平的显著增加相关。 总之，我们的研究结果链接的核心NER/TFIIH复合物和功能所需的同源重组/DNA双链断裂修复与基因组的稳定性，并通过涉及DNA降解的机制，对Ty1逆转录转座宿主防御。

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.