Regulation of telomere length

描述(申请人提供)：端粒是特殊的染色质结构，保护染色体末端，由富含甘油三酯的短序列重复序列和结合它们的蛋白质组成。维持重复序列需要端粒酶，这是一种酶-RNA复合体，其内部RNA包含合成新重复序列的模板，在具有端粒酶的细胞中，端粒长度被调节，以便细胞中最短的端粒优先被延长。在上一个资金周期中，我们发现细胞周期检查点激酶Tel1p优先被招募到短端粒中，其激酶活性是端粒延长所必需的。我们假设，Tel1p募集到短端粒至少部分是通过Rif1p的磷酸化来刺激端粒的伸长，Rif1p是端粒染色质的一种成分，也是已知的端粒长度的负调节因子。上一个周期的其他工作表明，Tel1p Paralog Mec1p具有与Tel1p不同的功能，因为当端粒缩短到功能障碍并导致细胞周期停滞时，它与端粒相关。我们假设Mec1p与功能失调的端粒相关，通过磷酸化Tbf1p刺激延伸，Tbf1p是一种与端粒重复序列相邻的DNA结合的蛋白质，参与了端粒伸长的调节。在前一个周期中，我们还开发了毒性的端粒酶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一种治疗高表达端粒酶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)通过测试Tel1p端粒长度控制涉及Rif1p磷酸化的假设(目标1)，2)通过确定控制Mec1p募集到端粒并允许Mec1p依赖的端粒延长的过程(目标2)，以及3)通过定义有毒的端粒酶RNA等位基因如何抑制细胞生长(目标3)。这些目的的成功完成将为我们理解真核生物的端粒长度调控做出实质性的贡献。与公共健康相关：端粒是染色体的物理末端，在染色体的维持和复制中发挥重要作用。端粒功能缺陷被认为与癌症和人类干细胞寿命缩短有关，从而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，我们将在这一应用中进行研究。了解酵母中的这些机制将为在人类细胞中测试类似的机制提供一个模型。

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.