Function and Dynamics of Tetrahymena Telomere Proteins

四膜虫端粒蛋白的功能和动态

• 批准号: 7903093
• 负责人: Carolyn M Price
• 金额: $ 31.09万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903093
• 关键词: Address; Affect; Affinity; Binding; Biochemical; Biochemistry; Caspase-1; Cell Cycle; Cell Cycle Regulation; Cells; Chromatin; Chromosomal Breaks; Chromosome Breakage; Chromosomes; Complex; DNA; DNA Damage; DNA Repair; DNA Sequence; DNA Sequence Rearrangement; DNA-Directed DNA Polymerase; Defect; Development; Digestion; Dyskeratosis Congenita; Ensure; Enzymes; Exhibits; Family member; Genes; Genetic; Global Change; Goals; Growth; Healed; Health; Hereditary Disease; Homologous Gene; Human; Individual; Knock-out; Label; Lead; Length; Malignant Neoplasms; Monitor; Nature; Organism; Outcome; Oxytricha; Partner in relationship; Phase; Phenotype; Play; Process; Proteins; Pulmonary Fibrosis; Recombinant DNA; Recruitment Activity; Regulation; Research; Role; Site; Specificity; Structure; Telomerase; Telomere Capping; Telomere Shortening; Testing; Tetrahymena; Transferase; Western Blotting; Work; chromosome replication; healing; human disease; novel; nuclease; prevent; protein complex; protein function; public health relevance; telomere; trait

DESCRIPTION (provided by applicant): The overall goal of this research is to understand how telomere proteins regulate telomerase recruitment and other the steps in telomere replication, but yet ensure that the chromosome terminus is protected from unwanted DNA repair or processing activities during the remainder of the cell cycle. The research is pertinent to human health because loss of telomere capping leads to chromosome fusions and chromosome rearrangements, the underlying cause of many forms of cancer. Defects in telomerase action are also associated with human disease as the resulting telomere shortening can lead to dyskeratosis congenita and pulmonary fibrosis. Telomere protection is generally achieved by multi-protein complexes that bind the telomeric DNA and sequester the DNA terminus. During replication, some of the same proteins regulate processing of the telomeric DNA, recruit telomerase, or stimulate telomerase activity. However, the mechanism by which individual proteins regulate these processes is still poorly understood. We will address this question through aims 1 and 3 which examine how three newly identified Tetrahymena telomere proteins, p61, p45 and Pot1b, regulate access to telomerase, enhance telomerase activity, or promote new telomere synthesis. Aim 2 addresses the dynamic nature of telomere structure. Telomeres must undergo a structural switch during S-phase in order to make the chromosome terminus accessible. Despite the importance of this structural change, the underlying cause is not understood. We will tackle this problem by examining Tetrahymena telomere composition and how this changes during the cell cycle or under different growth conditions. We will use Tetrahymena for our work because it has unique traits that make it exceptionally well suited for the genetic and biochemical approaches needed to address the above questions. In particular, it has ~40,000 telomeres per cell, correspondingly high levels of telomerase, and telomeres that exist as discrete complexes which can be purified from bulk chromatin. The specific aims are as follows. Aim 1: Determine the function of p61 and p45 in telomere capping and telomerase regulation. Aim 2: Characterize Tetrahymena telomere composition and dynamics. Aim 3: Determine the role of Pot1b during new telomere synthesis. The outcome should be a comprehensive picture of the role played by individual telomere proteins in telomere protection, replication, and chromosome healing, and of how the dynamic changes in telomere organization promote these processes. PUBLIC HEALTH RELEVANCE: Telomeres are the protective DNA-protein caps at chromosome ends that prevent chromosome fusions and degradation of the terminal DNA sequence. Proteins that make up the protective cap structure have to perform different activities as cells go through the cell cycle. During much of the cell cycle, they must hide the chromosome end so that it is not degraded or fused to another chromosome. However, when the chromosome is replicated they have to make the DNA terminus accessible and they play an active role in recruiting factors needed to replicate the telomeric DNA. The proposed research seeks to understand how telomere proteins function individually, and as part of the larger telomere protein complex, to achieve these opposing roles. It also investigates the function of a novel protein that seems to help broken chromosomes by promoting the addition of a new telomere. The research is pertinent to human health because loss of telomere protection leads to chromosome fusions and chromosome rearrangements, the underlying cause of many forms of cancer. Defects in telomerase, the enzyme that maintains telomere length, are also associated with human disease as the resulting telomere shortening can lead to dyskeratosis congenita and pulmonary fibrosis.

描述（由申请人提供）：本研究的总体目标是了解端粒蛋白如何调节端粒酶募集和端粒复制的其他步骤，但仍确保染色体末端在细胞周期的剩余时间内免受不必要的DNA修复或加工活动的保护。这项研究与人类健康有关，因为端粒盖的缺失会导致染色体融合和染色体重排，这是许多癌症的潜在原因。端粒酶作用的缺陷也与人类疾病有关，因为由此产生的端粒缩短可导致先天性角化不良和肺纤维化。端粒保护通常通过结合端粒DNA并隔离DNA末端的多蛋白复合物来实现。在复制过程中，一些相同的蛋白质调节端粒DNA的加工，招募端粒酶，或刺激端粒酶的活性。然而，单个蛋白质调节这些过程的机制仍然知之甚少。我们将通过目的1和3来解决这个问题，目的1和3研究三种新发现的四膜端粒蛋白p61， p45和Pot1b如何调节端粒酶的获取，增强端粒酶活性或促进新的端粒合成。目的2解决端粒结构的动态性质。端粒在s期必须经历结构转换，以使染色体末端可接近。尽管这种结构性变化很重要，但根本原因尚不清楚。我们将通过检查四膜体端粒组成以及在细胞周期或不同生长条件下端粒组成如何变化来解决这个问题。我们将在我们的工作中使用四膜虫，因为它具有独特的特性，使其非常适合解决上述问题所需的遗传和生化方法。特别是，它每个细胞约有40000个端粒，相应的端粒酶水平很高，端粒以离散的复合物形式存在，可以从大量染色质中纯化。具体目的如下。目的1：确定p61和p45在端粒盖帽和端粒酶调控中的功能。目的2：表征四膜虫端粒的组成和动态。目的3：确定Pot1b在新端粒合成中的作用。结果应该是一个全面的画面，在端粒保护，复制和染色体愈合中发挥作用的单个端粒蛋白，以及如何在端粒组织的动态变化促进这些过程。