MECHANISM OF ACTION OF YEAST SILENCING COMPLEXES

酵母沉默复合物的作用机制

• 批准号: 6636485
• 负责人: DANESH MOAZED
• 金额: $ 24.94万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636485
• 关键词: DNA binding protein; Saccharomyces cerevisiae; amidohydrolases; antibody; chromatin; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; fungal genetics; fungal proteins; gene induction /repression; histones; mass spectrometry; nicotinamide adenine dinucleotide; protein purification; protein reconstitution; ribosomal DNA

The long-term goal of this work is to understand in precise molecular terms how large DNA domains are packaged into an inactive chromatin structure called heterochromatin or silent chromatin. This type of chromatin plays a central role in the regulation of gene expression and maintenance of chromosome stability in organisms ranging from yeast to human. Studies in yeast have identified several gene products that are involved in the assembly of silent chromatin, and the types of complexes that these gene products form have been characterized. We have recently shown that a conserved component of silent chromatin, the Sir2 protein, has a functionally essential ADP-ribosyltransferase activity. This proposal will use purified silencing complexes, together with a chromatin assembly system, to reconstitute silent chromosome domains in vitro. This system will then provide the basis for biochemical experiments to deduce molecular mechanisms and the significance of the enzymatic activity of Sir2 in gene silencing. Silent chromatin is a highly conserved feature of eukaryotic chromosomes. It therefore seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply to other settings. A basic understanding of the molecular events that control chromosome structure and ensure the stable inheritance of cell fate provides not only a frame work for understanding how the process can fail, but also provides substrates and knowledge to design therapeutic strategies based on intervention.

这项工作的长期目标是在精确的分子术语中了解大的DNA结构域是如何包装成称为异染色质或沉默染色质的非活性染色质结构的。 这种类型的染色质在从酵母到人类的生物体中基因表达的调节和染色体稳定性的维持中起着核心作用。 对酵母的研究已经确定了参与沉默染色质组装的几种基因产物，并且这些基因产物形成的复合物类型已得到表征。 我们最近发现，沉默染色质的一个保守的组成部分，Sir 2蛋白，具有功能上必不可少的ADP-核糖基转移酶活性。 该建议将使用纯化的沉默复合物，与染色质组装系统一起，在体外重建沉默的染色体结构域。 该系统将为生物化学实验提供基础，以推断Sir 2的酶活性在基因沉默中的分子机制和意义。沉默染色质是真核生物染色体的一个高度保守的特征。 因此，本提案中为酵母蛋白质开发的许多原则可能适用于其他环境。 对控制染色体结构和确保细胞命运稳定遗传的分子事件的基本理解不仅为理解该过程如何失败提供了框架，而且还为设计基于干预的治疗策略提供了基础和知识。