Ty Element Retrotransposition in Saccharomyces cerevisiae

一年来，我们在以下方面取得了进展。尽管酿酒酵母Ty1逆转录转座子和逆转录病毒的进化距离很远，但它们在复制、整合和与宿主相互作用方面使用相似的策略。我们已经检查了环状Ty1 DNA的形成，这与逆转录病毒感染期间出现的死端环状产物类似。当整合存在缺陷时，存在明显水平的环状Ty1 DNA，其中一长末端重复序列(LTR)环和删除的环构成主要类别，而两个LTR环被丰富。当同源重组途径被突变阻断时，One-LTR环仍然存在，这表明它们是反转录的结果。Ty1自整合事件很容易发生，许多事件与DNA折叠结构重合，并依赖于DNA折叠结构，DNA折叠结构是由中央多尿路启动的DNA合成产生的。这些结果表明，Ty1特异性机制将拷贝数降至最低，并提高了特殊DNA结构是靶向决定因素的可能性。在与SAIC公司的乔纳森·凯勒的合作中，我们研究了p205的功能，它是干扰素诱导的调节细胞增殖的P200蛋白家族的成员。P205过表达可抑制细胞生长，但其作用机制目前尚不清楚。因此，我们评估了p205对P53和Rb依赖的细胞周期调节通路的影响。P205的表达导致p21水平升高，并在体外以p53依赖的方式激活p21启动子</I>。此外，p205还诱导Rb表达增加，并与Rb和P53直接结合。有趣的是，p205还通过延缓增殖细胞的G2/M进程而诱导不依赖于P53和Rb的生长抑制，并且是CDK2激酶活性的底物。最后，我们通过酵母双杂交筛选确定了p205的其他结合伙伴，包括配对的同源结构域蛋白HoxB2。综上所述，我们的结果表明，p205通过与调节细胞周期的多个转录因子相互作用而诱导生长停滞，包括但不完全依赖于Rb和P53介导的生长抑制途径。

Over the past year, we have made progress in the following areas. Despite their evolutionary distance, the <I>Saccharomyces cerevisiae</i> retrotransposon Ty1 and retroviruses use similar strategies for replication, integration, and interactions with their hosts. We have examined the formation of circular Ty1 DNA, which is comparable to the dead-end circular products that arise during retroviral infection. Appreciable levels of circular Ty1 DNA are present with one-long terminal repeat (LTR) circles and deleted circles comprising major classes, while two-LTR circles are enriched when integration is defective. One-LTR circles persist when homologous recombination pathways are blocked by mutation, suggesting that they result from reverse transcription. Ty1 autointegration events readily occur, and many are coincident with and dependent upon DNA flap structures that result from DNA synthesis initiated at the central polypurine tract. These results suggest that Ty1-specific mechanisms minimize copy number and raise the possibility that special DNA structures are a targeting determinant. In a collaboration with Jonathan Keller (SAIC, Inc), we have studied the function of p205, which is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we have evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter <I>in vitro</i> in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.