Cell Cycle Regulation of Yeast Telomerase Assembly and Function

酵母端粒酶组装和功能的细胞周期调控

• 批准号: 7612672
• 负责人: Katherine Louise Friedman
• 金额: $ 29.55万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7612672
• 关键词: Address; Affect; Amino Acids; Ants; Binding; Cell Cycle; Cell Cycle Regulation; Cell division; Cells; Chromosomes; Cis-Acting Sequence; Complex; DNA; DNA Sequence; DNA biosynthesis; DNA-Binding Proteins; Defect; Eating; Enzymes; Event; Expressed Sequence Tags; G1 Phase; Genome Stability; Goals; Growth; Human; Kinetics; Laboratories; Lead; Maps; Mediating; Mediator of activation protein; Modeling; Multienzyme Complexes; Mutation; Nature; Phase; Play; Process; Proteasome Inhibition; Proteins; Publishing; RNA; RNA-Directed DNA Polymerase; Recruitment Activity; Regulation; Regulatory Pathway; Resistance; Ribonucleoproteins; Role; Saccharomyces cerevisiae; Structure; Telomerase; Telomere Maintenance; Testing; Time; Trans-Activators; Work; Yeasts; anaphase-promoting complex; cell growth; design; genetic regulatory protein; in vivo; interest; multicatalytic endopeptidase complex; neoplastic cell; novel; prevent; protein protein interaction; research study; telomere; tool; tumor

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.

描述(申请人提供)：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，提高了人们对端粒酶作为化疗靶点的兴趣。在酿酒酵母中，端粒酶含有一个RNA模板(TLC1RNA)，一个逆转录酶(Est2p)，以及至少两个额外的蛋白质组分est1p和est3p。在S晚期，Est2p与端粒结合，而Est1p与端粒结合，与端粒延长相一致。P.I.S实验室之前的工作表明，在G1期，est1p的受控降解阻止了端粒酶复合体的组装。通过蛋白酶体抑制使Est1p在G1期稳定下来，促进了Est1p和Est3p与Est2p的结合，揭示了est1p在Est3p重新招募到端粒酶复合体中的一个新的作用。尽管具有催化活性，但在G1期组装的酶并不会延长端粒，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该酶。这里提出的实验旨在解决有关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，这些实验将有助于识别可能作为人类肿瘤细胞端粒酶失活靶点的调节事件的长期目标。 本研究的目的是(1)确定影响Est1p降解的调控途径(S)；(2)研究Est1p促进复杂组装的机制；以及(3)表征细胞中端粒酶组装和激活的动力学。这些实验将首次详细描述细胞进行DNA复制时端粒酶复合体中发生的蛋白质相互作用。将开发新的工具来研究在G1期限制端粒酶活性的机制(S)，并探索这一调控的功能相关性。此外，端粒酶组件的顺式作用突变和/或干扰端粒酶组装的反式作用调控途径的缺陷也将被识别。这项工作的目的是描述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。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，这是一种限制细胞分裂次数的现象。肿瘤细胞通常通过激活一种名为端粒酶的酶来绕过这一限制，端粒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，我们希望找到抗肿瘤治疗的新靶点。