Structure and Behaviour of Yeast Telomeres

描述（由申请人提供）：端粒，线性染色体的末端，是DNA复制的挑战。在缺乏专门的复制机制的情况下，DNA在每一轮DNA复制中都会从染色体末端丢失。在大多数真核生物中，端粒酶解决了末端复制问题，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，分别是EST1、EST2、EST3、TLC1和CDC13。这些基因中的任何一种缺乏都会导致端粒逐渐缩短，最终导致细胞死亡。此外，检查点激酶Tel1p或Mec1p都是端粒酶介导的端粒延长所必需的。酵母端粒酶受细胞周期和端粒长度的调控。该研究的总体目标是利用遗传、生化和细胞生物学方法的结合来阐明这两种类型调控的分子细节。酵母端粒酶仅在S/G2期后期延长端粒，尽管催化亚基Est2p在整个细胞周期的大部分时间都与端粒相关。Est1p和Est3p仅在端粒酶作用时（S/G2期晚期）与端粒相关。此外，端粒酶优先作用于短端粒，Est1p、Est2p和Tel1p优先结合短端粒。我们将确定Tel1p与短端粒的优先结合是否需要它们的优先延长，研究Tel1p如何被招募到短端粒，并确定Mec1p是否与tel1细胞中的短端粒结合。我们将研究负调控因子Rif1p和Rif2p对端粒酶募集的影响。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，核苷酸分辨率分析）的新检测方法，检测从端粒末端（而不是从内部缺失）缩短的端粒上Rif1p或Rif2p结合是否被耗尽。使用一种新的系统来产生一个单一的长端粒，我们将确定在长端粒上是否富集了Rif结合。如果观察到端粒长度的差异结合，我们将确定这种行为对端粒与端粒酶端粒结合的影响。Rif1p有多个候选的Tel1p磷酸化位点：将进行实验来测试Rif1p是否以端粒长度依赖的方式被Tel1p磷酸化。如果Rif1p是Tel1p的靶点，我们将评估其磷酸化对端粒酶募集的影响。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特性，比如它们结合DNA和RNA的能力，以及它们在体外对端粒酶活性的影响。这些体外方法将补充体内对Est1p、Est3p和Cdc13p的研究，包括使用NRA+ChIP来确定这些蛋白是否优先与那些实际延长的端粒相关。我们还将分离功能est3和est1等位基因，以深入了解这些蛋白质在端粒酶途径中的功能，并使用细胞生物学方法来验证端粒与外周细胞的关联将它们与端粒酶分离的假设。

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.