Cell Cycle Regulation of Yeast Telomerase Assembly and Function

描述（由申请人提供）：核糖核蛋白复合物，端粒酶，抵消DNA复制过程中末端序列的损失。端粒酶的不适当激活促进了人类肿瘤细胞的永生生长，提高了人们对端粒酶作为化疗靶点的兴趣。在酿酒酵母中，端粒酶包含一个RNA模板（TLC1 RNA），一个逆转录酶（Est2p），以及至少两个额外的蛋白质成分，Est1p和Est3p。Est2p组成端粒结合，而Est1p在S期晚期与端粒结合，与端粒延长一致。私家侦探以前的工作在G1期Est1p的调控降解阻止了端粒酶复合物的组装。G1期通过蛋白酶体抑制稳定Est1p，促进Est1p和Est3p与Est2p的结合，揭示了Est1p在募集Est3p到端粒酶复合体中的新作用。虽然具有催化活性，但G1期间组装的酶不会延长端粒，这表明当细胞进入S期时，必须发生额外的调节事件来激活酶。本文提出的实验旨在解决酵母细胞周期中端粒酶组装和激活机制的关键问题。由于Est2p和Est1p亚基在人类端粒酶中是保守的，这些实验将有助于确定人类肿瘤细胞中可能作为端粒酶失活靶点的调控事件的长期目标。

DESCRIPTION (provided by applicant): 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.