Regulation of telomere length

描述（由申请人提供）：端粒是保护染色体末端的特殊染色质结构，由富含tg的短序列重复序列和结合它们的蛋白质组成。重复序列的维持需要端粒酶，这是一种酶-RNA复合物，其内部RNA含有合成新重复序列的模板，在具有端粒酶的细胞中，端粒长度受到调节，因此细胞中最短的端粒优先被拉长。在之前的资助周期中，我们发现细胞周期检查点激酶Tel1p优先被募集到短端粒，并且其激酶活性是端粒延伸所必需的。我们假设，端粒短端粒的端粒1p募集至少在一定程度上是通过端粒染色质的组成部分Rif1p的磷酸化刺激了端粒长度的延长。先前周期的其他研究表明，与Tel1p相似的Mec1p与Tel1p具有不同的功能，因为当端粒缩短到功能失调并导致细胞周期停滞时，它与端粒有关。我们假设Mec1p与功能失调端粒的关联通过Tbf1p的磷酸化刺激了端粒延伸，Tbf1p是一种与端粒重复序列相邻的DNA结合的蛋白质，与端粒延伸的调节有关。在上一个周期中，我们还开发了毒性端粒酶RNA等位基因，这些等位基因极大地抑制了细胞的生长，这些等位基因代表了治疗端粒酶表达高水平的癌细胞的潜在疗法。我们的长期目标是了解端粒是如何维持的，以及它们如何完成其独特的细胞功能。我们将实现这些目标：1)通过验证端粒端粒长度控制涉及Rif1p磷酸化的假设（目的1），2)通过确定控制端粒端粒募集和允许依赖于Mec1p的端粒延长的过程（目的2），3)通过定义有毒端粒酶RNA等位基因如何抑制细胞生长（目的3）。这些目标的成功完成将对我们对真核生物端粒长度调控的理解做出重大贡献。公共卫生相关性：端粒是染色体的物理末端，在染色体的维持和复制中起重要作用。端粒功能缺陷与癌症和人类干细胞寿命缩短有关，降低了人体的修复能力。使用酵母作为人类细胞的模型，我们已经确定了端粒复制和端粒失效的关键步骤，我们将在本应用中进行研究。了解酵母中的这些机制将为在人类细胞中测试类似机制提供一个模型。

DESCRIPTION (provided by applicant): Telomeres are specialized chromatin structures that protect the chromosome end and are comprised of short TG-rich sequence repeats and the proteins that bind them. Maintenance of the repeats requires telomerase, an enzyme-RNA complex whose internal RNA contains a template for the synthesis of new repeats, and in cells that have telomerase, telomere length is regulated such that the shortest telomeres in the cell are preferentially elongated. In the previous funding cycle, we showed that the cell cycle checkpoint kinase Tel1p is preferentially recruited to short telomeres and that its kinase activity is required for telomere elongation. We hypothesize that Tel1p recruitment to short telomeres stimulates elongation, at least in part, through phosphorylation of Rif1p, a component of telomere chromatin and known negative regulator of telomere length. Other work in the previous cycle revealed that the Tel1p paralog Mec1p has a distinct function from Tel1p, as it associates with telomeres when they have shortened to the point where they become dysfunctional and result in cell cycle arrest. We hypothesize that Mec1p association with dysfunctional telomeres stimulates elongation through phosphorylation of Tbf1p, a protein bound to the DNA adjacent to telomere repeats that has been implicated in the regulation of telomere elongation. In the previous cycle, we also developed toxic telomerase RNA alleles that greatly inhibit cell growth, and these alleles represent a potential therapy to treat cancer cells that express high levels of telomerase. Our long-term goals are to understand how telomeres are maintained and how they accomplish their unique cellular functions. We will pursue these goals 1) by testing the hypothesis that Tel1p telomere length control involves Rif1p phosphorylation (Aim 1), 2) by determining the processes that govern Mec1p recruitment to telomeres and that allow Mec1p-dependent telomere elongation (Aim 2), and 3) by defining how the toxic telomerase RNA alleles inhibit cell growth (Aim 3). Successful completion of these aims will make substantial contributions to our understanding of telomere length regulation in eukaryotes. PUBLIC HEALTH RELEVANCE: Telomeres are the physical ends of chromosomes and play important roles in chromosome maintenance and replication. Defects in telomere function have been implicated in cancer and a decreased life span of human stem cells, decreasing the body's capacity for repair. Using yeast as a model for human cells, we have identified key steps in telomere replication and telomere failure that we will investigate in this application. Understanding these mechanisms in yeast will provide a model for testing similar mechanisms in human cells.