Regulation of retrotransposition in S.cerevisiae

描述（由申请人提供）：长末端重复(LTR)-逆转录转座子是类似逆转录病毒的可移动元件。由ltr -反转录转座子合成的RNA被复制回DNA，然后这种“复制DNA”（cDNA）被整合到基因组的新位置。ltr -反转录转座子广泛存在于真核生物中，它们激活和灭活宿主基因，促进基因组重排，产生和分散宿主基因的拷贝。因此，它们在真核生物基因组的结构和进化中起着重要的作用。酵母（Saccharomyces cerevisiae）的Ty1 ltr -反转录转座子具有典型的功能性和高水平表达，但由于cDNA合成受到许多宿主因子的抑制，转座子很少发生。我们工作的长期目标是了解功能性ltr -反转录转座子的休眠是如何维持的。我们最近的研究表明，端粒侵蚀在端粒酶缺失的情况下会触发“损伤诱导的Ty1激活途径”，这是一种调动Ty1元素的dna损伤信号通路。在本提案中，我们将验证这样一个假设，即在高等真核生物中，基因组稳定和抑制肿瘤形成所必需的一大群基因（称为看守基因）阻断了触发“病变诱导的Ty1激活途径”的DNA病变的形成。在缺乏它们的情况下，Ty1激活途径会刺激Ty1 cDNA的合成，从而导致过度转位。此外，提出的实验将验证端粒酶阴性突变体中Ty1逆转录酶活性增强导致称为Y'的亚端粒重复序列cDNA拷贝的产生和扩散的假设。Y重复序列的扩增是端粒酶不依赖、重组依赖的替代端粒结构的一个特征。另一种端粒结构允许酵母细胞和人类癌细胞在没有端粒酶的情况下持续生长。

DESCRIPTION (provided by applicant): Long terminal repeat (LTR)-retrotransposons are mobile elements that resemble retroviruses. RNA synthesized from LTR-retrotransposons is copied back into DNA, and this "copy DNA" (cDNA) is then integrated at a new location in the genome. LTR-retrotransposons are widespread throughout eukaryotes, where they activate and inactivate host genes, promote genome rearrangements, and produce and disperse copies of host genes. Hence, they play an important role in the structure and evolution of the eukaryotic genome. Ty1 LTR-retrotransposons of the yeast, Saccharomyces cerevisiae, are typically functional and expressed at high levels, yet transposition occurs rarely because cDNA synthesis is inhibited by numerous host factors. The long-term goal of our work is to understand how the dormancy of functional LTR-retrotransposons is maintained. Our recent work has demonstrated that telomere erosion in the absence of telomerase triggers the "lesion-induced Ty1 activation pathway", a DNA-damage signaling pathway that mobilizes Ty1 elements. In this proposal, we will test the hypothesis that a large group of genes necessary for genome stability and for the suppression of tumor formation in higher eukaryotes (known as caretaker genes) blocks the formation DNA lesions that trigger the "lesion-induced Ty1 activation pathway". In their absence, the Ty1 activation pathway stimulates the synthesis of Ty1 cDNA, which results in excessive transposition. Moreover, the proposed experiments will test the hypothesis that enhanced Ty1 reverse transcriptase activity in telomerase-negative mutants results in the production and dispersal of cDNA copies of a subtelomeric repeat called Y'. Amplification of Y' repeats is one characteristic of telomerase-independent, recombination-dependent alternative telomere structures. Alternative telomere structures allow both yeast cells and human cancer cells to grow continuously in the absence of telomerase. The specific aims are: 1. Screen previously identified inhibitors of Ty1 transposition for those that repress the lesion-induced Ty1 activation pathway. Identify and characterize the components of the lesion-induced Ty1 activation pathway. 2. Perform biochemical characterization of specific intermediates in retrotransposition in wild-type and mutant strains to identify the mechanism of stimulating cDNA synthesis in mutants in which the lesion-induced Ty1 activation pathway is activated. 3. Determine the structure of Y' cDNA in telomerase-negative mutants, and determine how and where Y' cDNA is inserted into the genome. Determine the role played by Ty1 gene products in the synthesis and mobility of Y' cDNA.