Function and Dynamics of Tetrahymena Telomere Proteins

描述(申请人提供)：这项研究的总体目标是了解端粒蛋白如何调节端粒酶的招募和端粒复制中的其他步骤，但又要确保染色体末端在细胞周期的其余部分不受不必要的DNA修复或处理活动的影响。这项研究与人类健康有关，因为端粒封顶的丢失会导致染色体融合和染色体重排，而染色体重排是许多形式癌症的根本原因。端粒酶活性缺陷也与人类疾病有关，因为端粒缩短会导致角化不良、先天性心脏病和肺纤维化。端粒保护通常是通过与端粒DNA结合并隔离DNA末端的多蛋白质复合体来实现的。在复制过程中，一些相同的蛋白质调节端粒DNA的处理，招募端粒酶，或刺激端粒酶活性。然而，单个蛋白质调节这些过程的机制仍然知之甚少。我们将通过目标1和目标3来解决这个问题，这两个目标研究了三个新发现的四膜虫端粒蛋白p61、p45和Pot1b是如何调节端粒酶的访问、增强端粒酶活性或促进新的端粒合成的。目的2探讨端粒结构的动态性质。在S期，端粒必须经历结构转换，才能使染色体末端可达。尽管这一结构性变化很重要，但其根本原因尚不清楚。我们将通过研究四膜虫端粒的组成以及在细胞周期或不同生长条件下端粒的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征，使其非常适合于解决上述问题所需的遗传和生化方法。特别是，它每个细胞有大约40,000个端粒，相应地端粒酶水平很高，并且端粒以离散的复合体存在，可以从大量染色质中提纯。具体目标如下。目的1：探讨p61和p45在端粒封顶和端粒酶调控中的作用。目的2：研究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。结果应该是一幅关于单个端粒蛋白在端粒保护、复制和染色体修复中所起作用的综合图景，以及端粒组织的动态变化如何促进这些过程。 与公共卫生相关：端粒是位于染色体末端的保护性DNA-蛋白质帽，防止染色体融合和末端DNA序列的降解。当细胞经历细胞周期时，组成保护帽结构的蛋白质必须执行不同的活动。在细胞周期的大部分时间里，它们必须隐藏染色体末端，这样它才不会降解或与另一条染色体融合。然而，当染色体复制时，它们必须使DNA末端可达，并在复制端粒DNA所需的招募因子中发挥积极作用。这项拟议的研究试图了解端粒蛋白如何单独发挥作用，以及作为更大的端粒蛋白复合体的一部分，实现这些相反的作用。它还研究了一种新的蛋白质的功能，这种蛋白质似乎通过促进新的端粒的增加来帮助断裂的染色体。这项研究与人类健康有关，因为端粒保护的丧失会导致染色体融合和染色体重排，这是许多形式癌症的根本原因。端粒酶是维持端粒长度的一种酶，端粒酶的缺陷也与人类疾病有关，因为端粒缩短会导致先天性角化不良和肺纤维化。

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.