Function and Dynamics of Tetrahymena Telomere Proteins

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。 以及端粒复制的其他步骤，但仍要确保染色体末端不受 在细胞周期的剩余时间内不需要的DNA修复或处理活动。这项研究是 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，许多形式癌症的根本原因。端粒酶作用缺陷 端粒缩短也与人类疾病有关，因为端粒缩短会导致角化不良 先天性心脏病和肺纤维化。端粒保护一般是通过多蛋白复合体实现的。 它结合端粒DNA并隔离DNA末端。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。我们将解决 这个问题是通过目标1和目标3来研究三个新发现的四膜虫端粒 蛋白质，p61，p45和Pot1b，调节对端粒酶的访问，增强端粒酶活性，或促进新的 端粒合成。目的2探讨端粒结构的动态性质。端粒必须经历一个 在S期进行结构转换，以使染色体末端可达。尽管 这一结构性变化的重要性，其根本原因尚不清楚。我们将通过以下方式解决这个问题 研究四膜虫端粒的组成及其在细胞周期和不同条件下的变化 生长条件。我们将在我们的工作中使用四膜虫，因为它有独特的特征使它 特别适合于解决上述问题所需的遗传和生化方法 问题。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 以离散的复合体形式存在的端粒，可以从大量染色质中提纯。具体目标是 具体如下。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。目标3：确定Pot1b的作用 在新的端粒合成期间。结果应该是一个全面的图景，展示了 单个端粒蛋白在端粒保护、复制和染色体修复中的作用，以及端粒蛋白如何 端粒结构的动态变化促进了这些过程。

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.