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丢失。端粒酶的不适当激活促进人类肿瘤细胞的永生化生长，提高了这种酶作为化疗靶点的兴趣。在酿酒酵母中，端粒酶包含RNA模板（TLC 1 RNA）、逆转录酶（Est 2 p）和至少两种额外的蛋白质组分Est 1 p和Est 3 p。Est 2 p与端粒组成性结合，而Est 1 p与S期晚期的端粒结合，与端粒延长一致。P.I.之前的工作的实验室证明，在G1期Est 1 p的调节降解阻止端粒酶复合物的组装。通过蛋白酶体抑制在G1期稳定Est 1 p促进Est 1 p和Est 3 p与Est 2 p的关联，揭示了Est 1 p在Est 3 p向端粒酶复合物的募集中的新作用。虽然具有催化活性，但在G1期组装的酶不会延长端粒，这表明当细胞过渡S期时，必须发生额外的调节事件才能激活酶。这里提出的实验旨在解决有关端粒酶组装和激活在酵母细胞周期中的机制的关键问题。由于Est 2 p和Est 1 p亚基在人端粒酶中是保守的，这些实验将有助于确定可能作为人肿瘤细胞端粒酶失活靶点的调控事件的长期目标。 本提案的目的是（1）确定影响Est 1 p降解的调控途径;（2）检查Est 1 p促进复合物组装的机制;（3）表征细胞中端粒酶组装和活化的动力学，这些细胞正在经历同步细胞周期。这些实验将首次详细描述细胞进行DNA复制时端粒酶复合物中发生的蛋白质相互作用。将开发新的工具来解决在G1期限制端粒酶活性的机制，并探索这种调节的功能相关性。此外，还将鉴定端粒酶组分中的顺式作用突变和/或干扰端粒酶组装的反式作用调节途径中的缺陷。这项工作的目标是描述在细胞周期中调节端粒酶的机制，并阐明这些事件对端粒酶活性和端粒维持的影响。普遍相关 在大多数人类细胞中，重复的细胞分裂会导致染色体缩短，这种现象限制了细胞分裂的次数。肿瘤细胞通常通过激活一种叫做端粒酶的酶来规避这种限制，这种酶能够取代复制过程中丢失的DNA序列。通过研究调节端粒酶活性的机制，我们希望确定抗肿瘤治疗的新靶点。

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.