Structure and Behaviour of Yeast Telomeres

描述(申请人提供)：端粒，线性染色体的末端，是DNA复制的一个挑战。在没有专门的复制机制的情况下，每一轮DNA复制都会从染色体末端丢失DNA。在大多数真核生物中，末端复制问题是由端粒酶解决的，端粒酶是一种端粒特异性逆转录酶，它利用端粒DNA的RNA成分作为模板来延伸端粒DNA的G链。酿酒酵母体内端粒酶的作用至少需要5个基因：EST1、EST2、EST3、TLC1和CDC13。这些基因中的任何一个的缺陷都会导致端粒的进行性缩短，最终导致细胞死亡。此外，端粒酶介导的端粒延长需要Tel1p或Mec1p两种检查点激酶之一。酵母端粒酶受细胞周期和端粒长度的调节。这项拟议研究的总体目标是结合遗传、生化和细胞生物学方法阐明这两种类型调控的分子细节。酵母端粒酶只在S/G2期晚期延长端粒，尽管催化亚单位Est2p在细胞周期的大部分时间都与端粒相关。Est1p和Est3p是仅在端粒酶作用时才相关的端粒，处于S/G2期晚期。此外，端粒酶在短端粒上优先发挥作用，而且Est1p、Est2p和Tel1p优先与短端粒结合。我们将确定是否需要Tel1p与短端粒的优先结合才能优先延长它们，研究Tel1p是如何被招募到短端粒的，并确定Mec1p是否与tel1细胞中的短端粒结合。负调控因子Rif1p和Rif2p对端粒酶招募的影响将被检测。我们将使用一种结合染色质免疫沉淀(CHIP)和单个端粒序列分析(NRA，核苷酸解析分析)的新方法，测试从末端缩短(而不是内部缺失)的端粒上的Rif1p或Rif2p结合是否耗尽。使用一种新的系统来产生单个长端粒，我们将确定Rif结合是否在长端粒上丰富。如果看到端粒长度的不同结合，我们将确定这一行为对Tel1p和端粒酶的端粒结合的影响。Rif1p有多个候选的Tel1p磷酸化位点：将进行实验，以测试Rif1p是否被Tel1p以端粒长度依赖的方式磷酸化。如果Rif1p是Tel1p的靶标，我们将评估其磷酸化对端粒酶招募的影响。我们最近在纯化Est1p、Est3p和CDc13p方面的成功将被用来确定这些蛋白质的性质，例如它们与DNA和RNA结合的能力，以及它们对体外端粒酶活性的影响。这些体外方法将补充对est1p、est3p和cdc13p的体内研究，包括使用NRA+芯片来确定这些蛋白质是否优先与实际上延长的端粒相关。我们还将分离Est3和Est1等位基因的功能，以深入了解这些蛋白质如何在端粒酶途径中发挥作用，并使用细胞生物学方法来验证端粒与外围结合将它们与端粒酶隔离的假说。 大多数人类体细胞不表达端粒酶，因此它们的端粒慢慢缩短。端粒过短会引发基因组不稳定，从而导致癌症或干细胞衰竭。大多数人类肿瘤，无论是哪种类型的肿瘤，都表达端粒酶，这种表达可能有助于它们的不受限制的生长。许多参与人类端粒长度调节的蛋白质首先在酵母中被发现，酵母仍然是理解人类酶的关键模型。

DESCRIPTION (provided by applicant): Telomeres, the ends of linear chromosomes, are a challenge for DNA replication. In the absence of a specialized replication mechanism, DNA is lost from chromosome ends with each round of DNA replication. In most eukaryotes, the end replication problem is solved by telomerase, a telomere specific reverse transcriptase that extends the G-strand of telomeric DNA using its RNA component as a template. Telomerase action in S. cerevisiae in vivo requires at least five genes, EST1, EST2, EST3, TLC1 and CDC13. Deficiencies in any of these genes lead to progressive telomere shortening and eventually cell death. In addition, either of the checkpoint kinases Tel1p or Mec1p is required for telomerase-mediated telomere lengthening. Yeast telomerase is regulated by both the cell cycle and telomere length. The overall goal of the proposed research is to elucidate the molecular details of both types of regulation using a combination of genetic, biochemical, and cell biological approaches. Yeast telomerase lengthens telomeres only in late S/G2 phase, even though the catalytic subunit, Est2p, is telomere associated throughout most of the cell cycle. Est1p and Est3p are telomere associated only at the time of telomerase action, late S/G2 phase. In addition, telomerase acts preferentially at short telomeres, and Est1p, Est2p and Tel1p bind preferentially to short telomeres. We will determine if preferential binding of Tel1p to short telomeres is required for their preferential lengthening, examine how Tel1p is recruited to short telomeres, and determine if Mec1p binds short telomeres in tel1 cells. The effects of the negative regulators Rif1p and Rif2p on telomerase recruitment will be examined. We will test if Rif1p or Rif2p binding is depleted at telomeres that are shortened from their ends (rather than from internal deletion) using a new assay that combines chromatin immuno-precipitation (ChIP) with sequence analysis of individual telomeres (NRA, nucleotide resolution assay). Using a new system to generate a single long telomere, we will determine if Rif binding is enriched at long telomeres. If differential binding by telomere length is seen, we will determine the consequences of this behavior for the telomere association of Tel1p and telomerase. Rif1p has multiple candidate Tel1p phosphorylation sites: experiments to test if Rif1p is phosphorylated by Tel1p in a telomere length dependent manner will be conducted. If Rif1p is a Tel1p target, we will assess the consequences of its phosphorylation on telomerase recruitment. Our recent success in purifying Est1p, Est3p, and Cdc13p will be exploited to determine the properties of these proteins, such as their ability to bind DNA and RNA, and their effects on telomerase activity in vitro. These in vitro approaches will complement in vivo studies on Est1p, Est3p and Cdc13p, including use of NRA+ChIP to determine if these proteins are preferentially associated with those telomeres that are actually lengthened. We will also isolate gain of function est3 and est1 alleles to obtain insights into how these proteins function in the telomerase pathway and use cell biological approaches to test the hypothesis that telomere association with the periphery sequesters them from telomerase. Most human somatic cells do not express telomerase, and as a result their telomeres slowly shorten. Short telomeres can initiate genome instability which can lead to cancer or stem cell failure. Most human tumors, regardless of tumor type, express telomerase, and this expression probably contributes to their unconstrained growth. Many of the proteins involved in telomere length regulation in humans were first identified in yeast, which continues to serve as key model for understanding the human enzyme.