Cell Cycle Regulation of Yeast Telomerase Assembly and Function

项目摘要 核糖核蛋白复合体端粒酶在DNA复制过程中抵消末端序列的丢失。 端粒酶的不适当激活促进了人类肿瘤细胞的永生生长，引起了人们的兴趣 酶作为化疗靶点。在酿酒酵母中，端粒酶含有一个RNA模板 (TLC1 RNA)，逆转录酶(Est2p)，以及至少两个额外的蛋白质组分，est1p和est3p。 Est2p是结构性端粒结合的，而Est1p在S晚期与端粒结合，这与 端粒延长。P.I.S实验室之前的工作表明，受调控的降解 Est1p在G1期阻止端粒酶复合体的组装。Est1p在G1期的稳定化 通过抑制蛋白酶体促进est1p和est3p与est2p的关联，揭示了Est2p的一个新作用 Est1p在募集Est3p到端粒酶复合体中。尽管具有催化活性，但酶组装在一起 在G1期间不会延长端粒，这表明必须发生额外的调节事件来激活端粒 酶作为细胞过渡的S期。这里提出的实验旨在解决关键问题 关于酵母细胞周期中端粒酶组装和激活的机制。因为Est2p 和est1p亚基在人类端粒酶中是保守的，这些实验将有助于实现 确定可能作为人类肿瘤细胞端粒酶失活靶点的调控事件。 这项建议的目的是(1)确定影响est1蛋白降解的调控途径(S)；(2) 研究est1p促进复杂组装的机制；以及(3)表征Est1p的动力学 处于同步细胞周期的细胞中端粒酶的组装和激活。这些实验将 首次详细描述了作为细胞的端粒酶复合体中发生的蛋白质相互作用 进行DNA复制。将开发新的工具来解决限制端粒酶的机制(S) 在G1期的活性，并探讨这一调控的功能相关性。此外，顺位代理 端粒酶成分突变和/或干扰反式作用调控通路的缺陷 端粒酶组装将被鉴定。这项工作的目标是描述调控机制 并阐明这些事件对端粒酶活性和端粒酶活性的影响 端粒维持。一般关联性 在大多数人类细胞中，反复的细胞分裂会导致染色体缩短，这种现象 限制细胞可以分裂的次数。肿瘤细胞通常通过激活一种 一种被称为端粒酶的酶，能够取代复制过程中丢失的DNA序列。通过检查 通过对端粒酶活性调控机制的研究，我们希望为抗肿瘤治疗寻找新的靶点。

Project Summary The ribonucleoprotein complex, telomerase, counteracts loss of terminal sequences during DNA replication. Inappropriate activation of telomerase facilitates immortal growth of human tumor cells, raising interest in this enzyme as a chemotherapeutic target. In Saccharomyces cerevisiae, telomerase contains an RNA template (TLC1 RNA), a reverse transcriptase (Est2p), and at least two additional protein components, Est1p and Est3p. Est2p is constitutively telomere-bound, while Est1p associates with telomeres in late S phase, coincident with telomere lengthening. Previous work from the P.I.'s laboratory demonstrated that the regulated degradation of Est1p during G1 phase prevents assembly of the telomerase complex. Stabilization of Est1p during G1 phase by proteasome inhibition promotes association of both Est1p and Est3p with Est2p, uncovering a novel role of Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, enzyme assembled during G1 does not lengthen telomeres, suggesting that additional regulatory events must occur to activate the enzyme as cells transit S phase. The experiments proposed here are designed to address critical questions regarding the mechanism of telomerase assembly and activation in the yeast cell cycle. Because the Est2p and Est1p subunits are conserved in human telomerase, these experiments will facilitate the long-term goal of identifying regulatory events that may serve as targets of telomerase inactivation in human tumor cells. Aims of this proposal are to (1) determine the regulatory pathway(s) that impinge upon Est1p degradation; (2) examine the mechanism through which Est1p facilitates complex assembly; and (3) characterize the kinetics of telomerase assembly and activation in cells transiting a synchronous cell cycle. These experiments will provide the first detailed description of protein interactions that occur in the telomerase complex as cells undergo DNA replication. Novel tools will be developed to address the mechanism(s) that restrict telomerase activity during G1 phase and to probe the functional relevance of this regulation. In addition, cis-acting mutations in telomerase components and/or defects in trans-acting regulatory pathways that perturb telomerase assembly will be identified. The goals of this work are to describe the mechanisms regulating telomerase during the cell cycle and to elucidate the consequences of these events for telomerase activity and telomere maintenance. General Relevance In most human cells, repeated rounds of cell division result in chromosome shortening, a phenomenon that restricts the number of times a cell can divide. Tumor cells often circumvent this limitation by activating an enzyme called telomerase that is capable of replacing DNA sequences lost during replication. By examining the mechanisms that regulate telomerase activity, we hope to identify novel targets for anti-tumor therapy.