Retrotransposition in Saccharomyces cerevisiae

我们的研究工作旨在了解Ty元件逆转录转座的机制和后果。反转录转座子是一类在结构和复制方式上与逆转录病毒(如HIV-1)相似的转座元件。在哺乳动物和植物基因组中，TY元素的亲缘关系也占有相当大的比例。TY元件是研究逆转录转座的许多方面的范例，因为这些元件存在于酿酒酵母中，这是一个高度发达的真核模型系统。 真核生物基因组含有潜在的不稳定序列，其重排会威胁到基因组的结构和功能。在这里，我们发现核苷酸切除修复(NER)/TFIIH解旋酶基因Rad3和SSL2(RAD25)的某些突变等位基因赋予合成致死性，并通过增加短序列重组和Ty1逆转录转座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的RNA或蛋白质水平，也不会显著改变靶点特异性。然而，这些突变导致断裂的DNA分子和未结合的Ty1基因的物理稳定性增加，从而导致更高水平的短序列重组和Ty1逆转录转座。我们还研究了细胞同源重组功能在Ty1逆转录转座中的作用。我们发现，在RAD52重组修复途径基因突变的细胞中，转座增加，但在专用于错配修复(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，RAD52组突变体中Ty1逆转录转座的增加与Ty1 cDNA水平的显著提高有关。总之，我们的结果将核心NER/TFIIH复合体的成分和同源重组/DNA双链断裂修复所需的功能与基因组稳定性联系起来，并通过一种涉及DNA降解的机制来防御Ty1逆转座。 我们已经证明，与逆转录病毒一样，Ty1病毒样颗粒(VLP)中的基因组RNA是二聚体的。Ty1二聚体也与逆转录病毒二聚体相似，因为它们在VLP的蛋白降解成熟过程中稳定下来。二聚体的稳定表明TyA1的其中一个切割产物具有核酸伴侣活性，因此在功能上与逆转录病毒核衣壳蛋白(NC)相当。有趣的是，逆转录病毒和Ty1二聚体的热稳定性相似，但对Ty1 RNA的285个碱基的顺式作用区域的研究表明，在辅助Ty1元件存在的情况下，该区域足以进行一些转座，但没有发现逆转录病毒RNA中存在亲吻环基序。此外，TyA1缺乏一个可识别的NC结构域，该结构域具有核酸伴侣活性。这些特征表明，Ty1 RNA的包装是通过一种独特的机制发生的。 Ty1逆转录病毒样移动遗传元件通过元件编码的整合酶(IN)蛋白转位到新的基因组位置。我们报道，纯化的重组IN足以将线性DNA正确整合到超螺旋靶质粒中。Ty1 VLP比IN更有效地整合供体DNA。VLP和IN介导的插入发生在目标的随机位置。镁比锰更适合正确的整合，而且两种阳离子都不能增强IN的非特异性核酸酶活性。Southern分析也得到了与正确整合事件一致的产物。重组IN和VLP利用许多但不是全部含有非Ty1末端的线性供体片段，包括已被证明在体内转座有缺陷的U3突变。综上所述，我们的结果表明，IN在体外足以整合Ty1，并且IN与外源供体的相互作用没有与内源元件那么严格。

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. 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. We have shown that, as in retroviruses, the genomic RNA in Ty1 virus-like particles (VLPs) is dimeric. The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the VLP. The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity, and therefore, is functionally equivalent to the retroviral nucleocapsid (NC) protein. Interestingly, the thermostability of retroviral and Ty1 dimers is similar, yet examination of a 285-base cis-acting region of Ty1 RNA that is evidently sufficient for some transposition in the presence of a helper Ty1 element, has not revealed a kissing loop motif present in retroviral RNA. Furthermore, TyA1 lacks a recognizable NC domain, which has nucleic acid chaperone activity. These features suggest that packaging of Ty1 RNA occurs by a unique mechanism. The Ty1 retrovirus-like mobile genetic element transposes to new genomic locations via the element-encoded integrase (IN) protein. We report that purified recombinant IN is sufficient for correct integration of a linear DNA into a supercoiled target plasmid. Ty1 VLPs integrate donor DNA more efficiently than IN. VLP and IN-mediated insertions occur at random sites in the target. Magnesium is preferred over manganese for correct integration, and neither cation enhances nonspecific nuclease activity of IN. Products consistent with correct integration events have also been obtained by Southern analysis. Recombinant IN and VLPs utilize many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo. Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.