Telomerase and Telomeres: Structure, Function and Regulation

描述(申请人提供)：端粒酶在大多数真核生物中是复制染色体末端所必需的，它是不寻常的，因为它由RNA和蛋白质亚基组成。这个项目的目标是更好地了解酵母和人类端粒酶的结构-功能关系，特别是通过其端粒底物的可及性来调节端粒酶的作用。了解端粒酶作用的基本原理对人类健康很重要，因为85%-90%的癌症涉及端粒酶的重新激活；因此，人们对抗端粒酶化疗药物和作为肿瘤疫苗抗原的TERT(端粒酶逆转录酶)非常感兴趣。在对酵母端粒酶的研究中，我们提出，RNA亚单位提供了一个柔性支架，将必要的蛋白质捆绑到复合体中。在特定的目标#1中，我们将在体外和体内对这一模型进行关键测试，并确定RNA的TERT(Est2p)结合区的结构和功能。在特定的目标#2中，我们将分析酵母调节亚基est1p的结构-功能关系，并确定其功能相互作用。我们将检验这一特定的假设，即est1p的N-末端区域是一个14-3-3磷酸氨基酸结合适配器，参与招募和激活端粒上的端粒酶。具体目标#3利用我们最近与端粒单链DNA结合的染色体封顶蛋白hPOT1的晶体结构，将这项工作扩展到人类系统。我们将检验这一假设，即hPOT1在一种由翻译后修饰和/或与人EST1蛋白相互作用驱动的“染色质重塑”反应中，从端粒酶抑制模式切换到易用性结合模式。

DESCRIPTION (provided by applicant): Telomerase, required for the replication of chromosome ends in most eukaryotes, is unusual in that it is comprised of RNA as well as protein subunits. The goal of this project is to better understand structure- function relationships in yeast and human telomerases, with special attention to the regulation of telomerase action via the accessibility of its telomeric substrate. Understanding the fundamentals of telomerase action is important for human health, as 85 - 90% of cancers involve reactivation of telomerase; thus, there is substantial interest in anti-telomerase chemotherapeutic agents and in TERT (telomerase reverse transcriptase) as an antigen for cancer vaccines. In studies of yeast telomerase, we have proposed that the RNA subunit provides a flexible scaffold to tether essential proteins into the complex. In specific aim #1, we will critically test this model in vitro and in vivo, and determine the structure and function of the TERT (Est2p)-binding region of the RNA. In specific aim #2, we will analyze structure-function relationships of the yeast regulatory subunit Est1p and determine its functional interactions. We will test the specific hypothesis that the N-terminal region of Est1p is a 14-3-3 phosphoaminoacid-binding adapter involved in recruiting and activating telomerase at the telomere. Specific aim #3 extends this work to the human system, taking advantage of our recent crystal structure of the chromosome-capping protein hPOT1 bound to telomeric single-stranded DNA. We will test the hypothesis that hPOT1 is switched from telomerase-inhibitory to faciliatory binding modes in a sort of "chromatin remodeling" reaction, driven by post-translational modification and/or interaction with human EST1 proteins.