Telomerase and Telomeres: Structure, Function and Regulation

描述（由申请人提供）：端粒酶是大多数真核生物染色体末端复制所必需的，它是不寻常的，因为它由RNA和蛋白质亚基组成。该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，特别关注端粒酶通过其端粒底物的可及性对端粒酶作用的调节。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再激活；因此，人们对抗端粒酶化疗药物和端粒酶逆转录酶作为癌症疫苗抗原有很大的兴趣。在酵母端粒酶的研究中，我们提出RNA亚基提供了一个灵活的支架，将必需的蛋白质连接到复合物中。在具体目标#1中，我们将在体外和体内对该模型进行严格测试，并确定RNA的TERT （Est2p）结合区域的结构和功能。在具体目标#2中，我们将分析酵母调控亚基Est1p的结构-功能关系，并确定其功能相互作用。我们将验证特定假设，即Est1p的n端区域是一个14-3-3磷酸结合适配器，参与端粒端粒酶的招募和激活。具体目标#3将这项工作扩展到人类系统，利用我们最近的染色体覆盖蛋白hPOT1与端粒单链DNA结合的晶体结构。我们将验证hPOT1在翻译后修饰和/或与人类EST1蛋白相互作用的驱动下，在一种“染色质重塑”反应中从端粒酶抑制模式切换到促进结合模式的假设。

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.