Structure and Behavior of Yeast Telomeres

描述（由申请人提供）： 端粒是真核生物染色体的末端，对基因组的完整性至关重要。它们保护染色体免于降解和融合，并作为基因表达的专门位点。细胞区分端粒和双链断裂，尽管矛盾的是，这两种结构有很多共同之处。由于传统的复制装置不能复制线性分子的末端，在大多数真核生物中，端粒DNA由一种专门的逆转录酶端粒酶维持。这项资助的长期目标是阐明端粒实现其各种功能的机制，利用S。cerevisiae，在较小程度上，S. pombe作为模型。在此资助期间的实验将集中在细胞周期和端粒长度调节端粒酶的分子基础。第一个目标是使用体内和体外方法的组合来确定稳定的端粒酶组装和DNA末端活性的要求。具体来说，我们将测试是否需要长的单链TG 1 -3尾来形成稳定的全酶-端粒复合物，以及端粒结合蛋白和/或端粒酶组分中的某些突变是否在这一步骤中有缺陷。为了确定Est 1和Est 3的确切作用，这两种端粒酶辅助蛋白在体内是必需的，但在体外不是必需的，我们将开发更多的生理体外试验来确定Est 1和Est 3的作用。 如果两者合作克服Cdc 13对端粒酶延伸的抑制。目的2：研究端粒结合蛋白Rif 1对端粒酶活性的顺式抑制作用。我们将测试两个假设来解释为什么Rif 1的丢失会导致端粒过度伸长。首先，Rif 1 抑制端粒酶与端粒的结合，并且一旦端粒变得足够短以成为端粒酶的优选底物，这种抑制通过其翻译后修饰而减轻。生物化学和遗传学的方法，提出了确定这些修改。这些研究将产生一组rif 1等位基因，这将是有用的Ri 1的未来功能研究，无论其作用模式。第二个模型提出，Rif 1通过端粒DNA促进复制叉的进展，在其缺失的情况下，叉停滞并经常断裂，这些断裂被端粒酶延长。通过测量DNA聚合酶占有率和在Rif 1缺失细胞中用端粒DNA的二维凝胶可视化复制中间体来监测复制叉进展。如果在不存在Rif 1的情况下检测到停顿增加，则遗传测定将确定这种停顿是否与断裂和端粒酶介导的伸长相关。Aim 3描述了一种蛋白质组学方法的延续，该方法使用质谱（MS）来鉴定新的端粒酶相互作用蛋白和这些蛋白质的修饰。通过多个端粒酶亚单位的同时过表达和表位标记，我们在两种S。cerevisiae和S. pombe和使用MS鉴定多种相关蛋白，其中几种已知影响端粒长度。这些新的蛋白质和蛋白质修饰将通过遗传学和生物化学的结合来研究，以确定它们在端粒酶过程中的确切作用。

DESCRIPTION (provided by applicant): Telomeres, the ends of eukaryotic chromosomes, are essential for genome integrity. They protect chromosomes from degradation and fusions and serve as specialized sites for gene expression. Cells distinguish telomeres from double strand breaks even though, paradoxically, the two structures have much in common. Because the conventional replication apparatus cannot duplicate the very ends of linear molecules, in most eukaryotes, telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The long term goal of this grant is to elucidate mechanisms that enable telomeres to fulfill their varied functions, using S. cerevisiae, and to a lesser extent, S. pombe as models. Experiments in this funding period will focus on the molecular basis of both cell cycle and telomere length regulation of telomerase. The first aim uses a combination of in vivo and in vitro approaches to determine requirements for stable telomerase assembly and activity on DNA ends. Specifically, we will test if long single-strand TG1-3 tails are required to form a stable holoenzyme-telomere complex and if certain mutations in telomere binding proteins and/or telomerase components are defective in this step. To determine the precise roles of Est1 and Est3, two telomerase accessory proteins that are essential in vivo but not in vitro, we will develop more physiological in vitro assays to determine if the two cooperate to overcome Cdc13 inhibition of telomerase elongation. Aim 2 focuses on Rif1, a telomere binding protein that inhibits telomerase activity in cis. We will test two hypotheses to explain why loss of Rif1 results in telomere hyper-elongation. The first is that Rif1 inhibits telomerase binding to telomeres, and this inhibition is relieved by its post translation modification once telomeres become short enough to be preferred substrates for telomerase. Biochemical and genetic approaches are proposed to identify these modifications. These studies will generate a set of rif1 alleles that will be useful for future functional studies on Ri1, regardless of its mode of action. The second model proposes that Rif1 promotes replication fork progression through telomeric DNA, and in its absence, forks stall and often break, and these breaks are lengthened by telomerase. Replication fork progression will be monitored by measuring DNA polymerase occupancy and by visualizing replication intermediates with two dimensional gels of telomeric DNA in Rif1-depleted cells. If increased pausing is detected in the absence of Rif1, a genetic assay will determine if this pausing is associated with breakage and telomerase-mediated elongation. Aim3 describes the continuation of a proteomics approach that uses mass spectrometry (MS) to identify novel telomerase interacting proteins and modifications of these proteins. By simultaneous over-expression and epitope tagging of multiple telomerase subunits, we isolated active telomerase in both S. cerevisiae and S. pombe and used MS to identify multiple associated proteins, several of which are known to affect telomere length. These new proteins and protein modifications will be studied by a combination of genetics and biochemistry to determine their exact roles in the telomerase process.