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（DSBs）的威胁，如果不进行修复，可能导致永久性细胞周期阻滞或死亡。因此，存在复杂的机制来有效地检测和修复由dsb产生的DNA末端。DNA末端也会出现在自然染色体末端，相反，为了保持基因组的完整性，必须保护这些末端不受DSB修复活动的影响，如非同源末端连接（NHEJ）。这是通过被称为端粒的特殊核蛋白结构实现的。现在很清楚，许多响应DNA dsb的活性也在正常端粒结构、功能和维护中起作用。其中一种蛋白质是Ku异二聚体，这是一种高亲和力的DNA末端结合复合物，对NHEJ至关重要，值得注意的是，端粒生物学的多个方面，如保护端粒免受异常修复活动的影响，端粒长度的调节，以及抑制端粒染色质结构的形成，这导致附近基因的转录沉默，称为端粒沉默。矛盾的是，Ku也是灾难性端到端融合的主要效应者，这种融合可能发生在功能失调的端粒上。Ku的NHEJ活性如何在野生型端粒中被抑制仍然不清楚。PI和其他人先前的工作已经确定Ku在dsb和端粒中具有不同的活性，但是这些位点的作用机制尚未完全阐明。最近，PI及其同事开发了Ku在dsb和端粒中的功能的“双面”模型，其中有一个向外的面，面向DNA末端，介导NHEJ，以及一个向内的面，当与端粒结合时，面向端粒染色质，介导端粒功能。这项工作的总体目标是阐明模式生物酿酒酵母端粒中Ku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)通过位点定向诱变进一步定义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的一个或多个端粒活性是否需要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中的作用。Specific Aim 2将使用遗传和生化方法鉴定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。特异性目标3将通过识别与nhej特异性表面1-螺旋相互作用的因子，进一步定义Ku的修复特异性外表面的功能；这些将包括NHEJ因子以及可能在端粒抑制ku介导的NHEJ的端粒因子。因此，通过遗传和分子方法的结合，本提案为该领域目前对Ku功能的理解做出了重大贡献，这可能会为人类细胞的研究提供信息，其中Ku是必不可少的。