Ty Element Retrotransposition in Saccharomyces cerevisiae

酿酒酵母中的 Ty 元件逆转录转座

• 批准号: 7592673
• 负责人: David J. Garfinkel
• 金额: $ 133.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592673
• 关键词: Area; Binding; Biology; Biomedical Research; CDKN1A gene; Cancerous; Cell Cycle; Cell Cycle Regulation Pathway; Cell Proliferation; Cells; Chromatin; Chromosome Deletion; Class; Collaborations; Complementary DNA; Complex; Coupled; Cytoplasm; DNA; DNA Polymerase I; DNA Sequence Rearrangement; DNA Structure; DNA biosynthesis; DNA chemical synthesis; Elements; Event; Family; Fungal Genome; Gagging; Genes; Genome; Goals; Growth; HIV; Homeodomain Proteins; Human; Human Genome; In Vitro; Indium; Infection; Integrase; Interferons; Learning; Length; Long Terminal Repeats; Malignant Neoplasms; Mediating; Modeling; Molecular; Mutate; Mutation; Nuclear Envelope; Numbers; Organism; Pathway interactions; Phosphotransferases; Process; Proliferating; Protein Family; Proteins; RNA; RNA Interference Pathway; RNA Polymerase III; RNA Polymerase III Transcription Pathway; RNA-Directed DNA Polymerase; Research; Retroelements; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; Saccharomyces cerevisiae; Saccharomycetales; Short Interspersed Nucleotide Elements; Site; Structure; Surgical Flaps; TP53 gene; Transcript; Translations; Virus-like particle; Yeasts; cell growth; functional genomics; genome sequencing; homologous recombination; human disease; interest; member; oncoprotein p21; particle; preference; promoter; transcription factor; yeast protein; yeast two hybrid system

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.

一年来，我们在以下方面取得了进展：尽管进化距离较远，酿酒酵母（Saccharomyces cerevisiae）逆转录转座子Ty1和逆转录病毒使用相似的策略进行复制、整合和与宿主的相互作用。我们已经检查了环状Ty1 DNA的形成，它与逆转录病毒感染期间产生的死端环状产物相当。环状Ty1 DNA存在相当水平的单链末端重复（LTR）环和缺失环，包括主要类别，而双链LTR环在整合缺陷时富集。当同源重组途径被突变阻断时，1 - ltr环仍然存在，这表明它们是由逆转录引起的。Ty1自整合事件很容易发生，并且许多与DNA瓣结构一致并依赖于DNA瓣结构，这是由中央多嘌呤管启动的DNA合成引起的。这些结果表明，ty1特异性机制最大限度地减少了拷贝数，并提高了特殊DNA结构是靶向决定因素的可能性。在与Jonathan Keller （SAIC, Inc）的合作中，我们研究了p205的功能，它是干扰素诱导的p200蛋白家族的一员，可以调节细胞增殖。p205的过表达抑制细胞生长，尽管其作用机制目前尚不清楚。因此，我们评估了p205对p53和rb依赖的细胞周期调控途径的影响。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> </ I>。此外，p205诱导Rb表达增加，并直接与Rb和p53结合。有趣的是，p205还通过延缓增殖细胞的G2/M进程，诱导独立于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鉴定了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，我们的研究结果表明，p205通过与多种调节细胞周期的转录因子相互作用诱导生长停滞，包括但不完全依赖于Rb-和p53介导的生长抑制途径。