Structural and Biochemical Analysis of Sir3

Sir3 的结构和生化分析

• 批准号: 6798236
• 负责人: BRIAN E HALL
• 金额: $ 4.73万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6798236
• 关键词: DNA repair; DNA replication; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; X ray crystallography; gene expression; gene induction /repression; heterochromatin; high performance liquid chromatography; histones; postdoctoral investigator; protein binding; protein protein interaction; protein purification; protein structure function; proteolysis; site directed mutagenesis; structural biology

DESCRIPTION (provided by applicant): Gene silencing in Saccharomyces cerevisiae has served as a model system for the study of heterochromatin formation in more complex organisms. Regulation of heterochromatin is critical for transcriptional repression, epigenetic inheritance, and the maintenance of genomic stability. In yeast, heterochromatin is known as silent chromatin and is formed by the distinct steps of nucleation and spreading through the concerted action of multiple proteins including Sir2, Sir3, and Sir4. Currently, a large body of genetic and biochemical data indicates that Sir3 is recruited to sites of silencing nucleation by Sir4 and mediates the spreading of silent chromatin to distal chromosomal regions through interactions with the N-terminal tails of histones H4 and H3. The goals of the proposed study are to define the structural basis for Sir3 action and to clarify the mechanisms of Sir3 recruitment and Sir3 induced spreading during the formation of silent chromatin. This will be accomplished by determining the crystal structures of Sir3 in complex with a histone H4 peptide and in complex with a Sir4 peptide. In addition, these interactions will be quantitatively characterized using biochemical and biophysical methods. Following structure determination, site directed mutagenesis studies are planned to identify critical residues and isolate mutants that can be used to more clearly define the role of Sir3 in vivo. The proposed work combined with existing data and work currently being undertaken at Harvard Medical School will establish a remarkably complete picture of how silent chromatin is formed in yeast, thereby providing a framework for the study heterochromatin in more complex organisms.

描述（由申请人提供）：酿酒酵母中的基因沉默已成为研究更复杂的生物中异染色质形成的模型系统。异染色质的调节对于转录抑制，表观遗传遗传和基因组稳定性的维持至关重要。在酵母中，异染色质被称为静音染色质，由成核的不同步骤形成，并通过包括SIR2，SIR3和SIR4在内的多种蛋白质的协同作用扩散。目前，大量的遗传和生化数据表明，SIR3被SIR4募集到沉默的成核部位，并通过与组蛋白H4和H3的N末端尾巴相互作用，介导静音染色质到远端染色体区域的扩散。拟议的研究的目标是定义SIR3作用的结构基础，并阐明SIR3募集的机制和SiR3在静音染色质形成过程中引起的散布。这将通过确定与组蛋白H4肽复合物中的Sir3的晶体结构以及与Sir4肽复合物中的结构来实现。另外，这些相互作用将使用生化和生物物理方法进行定量表征。按照结构确定，计划定向诱变研究以识别可用于更清楚地定义sir3在体内作用的关键残基和分离突变体。拟议的工作结合了哈佛医学院目前正在进行的现有数据和目前的工作，将为酵母中的静音染色质形成，从而为研究异染色质提供了一个更复杂的生物体中的框架。