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。在没有专门的复制机制的情况下，DNA在每一轮DNA复制中从染色体末端丢失。在大多数真核生物中，末端复制问题由端粒酶解决，端粒酶是一种端粒特异性逆转录酶，其使用其RNA组分作为模板延伸端粒DNA的G链。S.酿酒酵母在体内需要至少五个基因，EST 1、EST 2、EST 3、TLC 1和CDC 13。这些基因中的任何一个缺陷都会导致端粒的不断缩短，最终导致细胞死亡。此外，检查点激酶Tel 1 p或Mec 1 p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节。拟议的研究的总体目标是阐明这两种类型的调节使用遗传，生物化学和细胞生物学方法的组合的分子细节。酵母端粒酶仅在S/G2期晚期延长端粒，即使催化亚基Est 2 p在整个细胞周期的大部分时间内与端粒相关。Est 1 p和Est 3 p仅在端粒酶作用时，即S/G2晚期与端粒相关。此外，端粒酶优先作用于短端粒，并且Est 1 p、Est 2 p和Tel 1 p优先结合短端粒。我们将确定Tel 1 p与短端粒的优先结合是否是其优先延长所需的，研究Tel 1 p如何被招募到短端粒，并确定Mec 1 p是否与tel 1细胞中的短端粒结合。将检查负调节剂Rif 1 p和Rif 2 p对端粒酶募集的影响。我们将使用一种将染色质免疫沉淀（ChIP）与单个端粒序列分析（NRA）相结合的新检测方法来测试Rif 1 p或Rif 2 p结合是否在从末端缩短（而不是内部缺失）的端粒处耗尽。，核苷酸解析测定）。使用一个新的系统来产生一个单一的长端粒，我们将确定是否Rif结合富集在长端粒。如果发现端粒长度的差异结合，我们将确定Tel 1 p和端粒酶的端粒关联的这种行为的后果。Rif 1 p具有多个候选Tel 1 p磷酸化位点：将进行实验以测试Rif 1 p是否以端粒长度依赖性方式被Tel 1 p磷酸化。如果Rif 1 p是Tel 1 p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Est 1 p，Est 3 p和Cdc 13 p的成功将被利用来确定这些蛋白质的特性，如它们结合DNA和RNA的能力，以及它们对端粒酶活性的影响。这些体外方法将补充对Est 1 p，Est 3 p和Cdc 13 p的体内研究，包括使用NRA+ChIP来确定这些蛋白质是否优先与实际延长的端粒相关。我们还将分离功能性增益的est 3和est 1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测试端粒与外周结合将其与端粒酶隔离的假设。 大多数人体细胞不表达端粒酶，因此它们的端粒会慢慢缩短。短端粒可以引发基因组不稳定，这可能导致癌症或干细胞衰竭。大多数人类肿瘤，无论肿瘤类型，表达端粒酶，这种表达可能有助于其不受约束的生长。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.