The Roles of the Ku Heterodimer in Yeast Telomere Function

Ku 异二聚体在酵母端粒功能中的作用

• 批准号: 7371851
• 负责人: Alison A Bertuch
• 金额: $ 28.87万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-21 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371851
• 关键词: Address; Affect; Affinity; Affinity Chromatography; Animal Model; Binding; Biochemical Genetics; Biology; Cell Cycle Arrest; Cells; Cessation of life; Chromatin; Chromatin Structure; Chromosomes; Classification; Complex; DNA; DNA Binding; DNA Double Strand Break; DNA Repair; Data; Detection; Double Strand Break Repair; Eukaryota; Eukaryotic Cell; Excision; Face; G22P1 gene; Generations; Genes; Genome; Goals; Helix (Snails); Heterochromatin; Human; Insertional Mutagenesis; Ku Protein; Lead; Left; Length; Light; Maintenance; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; Molecular Genetics; Mutagenesis; Mutate; Mutation; Nucleoproteins; Organism; Phenocopy; Property; Proteins; Proteome; Publishing; Regulation; Reporting; Role; Saccharomyces cerevisiae; Site; Site-Directed Mutagenesis; Specific qualifier value; Structure; Surface; Telomerase; Testing; Work; XRCC5 gene; Yeasts; defined contribution; ear helix; gene repression; genetic analysis; mutant; novel; prevent; protein function; repaired; research study; response; telomere

DESCRIPTION (provided by applicant): Genome integrity is threatened by DNA double strand breaks (DSBs), which, if left unrepaired, can lead to permanent cell cycle arrest or death. Consequently, complex mechanisms exist for the efficient detection and repair of DNA ends created by DSBs. DNA ends are also encountered at natural chromosome termini, which, conversely, must be protected from DSB repair activities, such as nonhomologous end joining (NHEJ), in order to preserve genome integrity. This is achieved through the specialized nucleoprotein structures known as telomeres. It is now clear that many of the activities that function in response to DNA DSBs also function in normal telomere structure, function, and maintenance. One such protein is the Ku heterodimer, a high affinity DNA end binding complex crucial for NHEJ and, notably, multiple aspects of telomere biology, such as the protection of telomeres from aberrant repair activities, the regulation of telomere length, and the formation of a repressive telomeric chromatin structure, which results in the transcriptional silencing of nearby genes, known as telomeric silencing. Paradoxically, Ku is also a principal effector of the catastrophic end-to-end fusions that can occur at dysfunctional telomeres. How Ku's NHEJ activity is inhibited at wild type telomeres remains poorly defined. Previous work by the PI and others has firmly established that Ku performs distinct activities at DSBs vs. telomeres, however the mechanisms of action at these sites have yet to be fully elucidated. Recently, the PI and co-workers have developed a `two-face' model for Ku's functions at DSBs and telomeres, in which there is an outward face, oriented toward the DNA terminus, which mediates NHEJ, and an inward face, oriented toward telomeric chromatin when bound to a telomere, which mediates telomeric functions. The overall goal of the proposed work is to elucidate the molecular determinants of Ku's activities at telomeres in the model organism, Saccharomyces cerevisiae, thereby expanding and testing the two-face model. Specific Aim 1 will a) further define Ku's inward face, particularly with respect to Ku's telomere end protection property, via site-directed mutagenesis; b) determine whether one or more of Ku's telomeric activities require DNA end binding by generating and characterizing DNA end binding defective Ku proteins; and 3) determine the role of end binding in protecting broken as compared to telomeric ends by analyzing the properties of Ku mutants consisting of solely the DNA binding core. Specific Aim 2 will identify and characterize proteins that interact with Ku in telomere end protection or other telomeric functions using genetic and biochemical approaches. Specific Aim 3 will further define the function at Ku's repair-specific outward face by identifying the factor(s) that interact with an NHEJ-specific surface 1-helix it contains; these will include NHEJ-factors as well as telomeric factors that may inhibit Ku-mediated NHEJ at telomeres. Thus, through a combination of genetic and molecular approaches, this proposal offers to make a substantial contribution to the field's current understanding of the function of Ku, which may inform studies in human cells, where Ku is essential.

描述（由申请人提供）：基因组完整性受到 DNA 双链断裂 (DSB) 的威胁，如果不修复，可能导致永久性细胞周期停滞或死亡。因此，存在复杂的机制来有效检测和修复 DSB 产生的 DNA 末端。 DNA 末端也会在天然染色体末端遇到，相反，必须保护天然染色体末端免受 DSB 修复活动的影响，例如非同源末端连接 (NHEJ)，以保持基因组完整性。这是通过称为端粒的特殊核蛋白结构实现的。现在已经清楚，许多响应 DNA DSB 的活动也在正常端粒结构、功能和维护中发挥作用。其中一种蛋白质是 Ku 异二聚体，它是一种高亲和力 DNA 末端结合复合物，对于 NHEJ 以及端粒生物学的多个方面至关重要，例如保护端粒免受异常修复活动、端粒长度的调节以及端粒长度的形成。抑制性端粒染色质结构，导致附近基因的转录沉默，称为端粒沉默。矛盾的是，Ku 也是端粒功能失调时可能发生的灾难性端到端融合的主要效应器。 Ku 的 NHEJ 活性如何在野生型端粒上受到抑制仍不清楚。 PI 和其他人之前的工作已经确定 Ku 在 DSB 和端粒上执行不同的活动，但是这些位点的作用机制尚未完全阐明。最近，PI和他的同事们为Ku在DSB和端粒上的功能开发了一个“双面”模型，其中一个向外的面，面向DNA末端，介导NHEJ，另一个向内的面，面向介导NHEJ的DNA末端。端粒染色质与端粒结合，介导端粒功能。这项工作的总体目标是阐明模型生物酿酒酵母中 Ku 端粒活性的分子决定因素，从而扩展和测试双面模型。具体目标 1 将 a) 通过定点诱变进一步定义 Ku 的内向面，特别是关于 Ku 的端粒末端保护特性； b) 通过生成和表征 DNA 末端结合缺陷 Ku 蛋白，确定 Ku 的一种或多种端粒活性是否需要 DNA 末端结合； 3) 通过分析仅由 DNA 结合核心组成的 Ku 突变体的特性，确定末端结合与端粒末端相比在保护断裂方面的作用。具体目标 2 将使用遗传和生化方法来识别和表征在端粒末端保护或其他端粒功能中与 Ku 相互作用的蛋白质。具体目标 3 将通过识别与 NHEJ 特定表面 1 螺旋相互作用的因子，进一步定义 Ku 修复特定外表面的功能；这些将包括 NHEJ 因子以及可能抑制端粒处 Ku 介导的 NHEJ 的端粒因子。因此，通过遗传和分子方法的结合，该提案为该领域目前对 Ku 功能的理解做出了重大贡献，这可能为人类细胞的研究提供信息，而 Ku 在人类细胞中至关重要。