STRUCTURE & BEHAVIOR OF YEAST TELOMERES

端粒是真核细胞染色体的末端，对 酵母菌的完全复制和稳定维持 染色体。端粒保护染色体不会退化，有助于 细胞区分完整的DNA和断裂的DNA，为 端粒酶，并影响附近基因的转录。整体而言 该项目的目标是识别相互作用的结构蛋白。 并确定它们对端粒的贡献 功能。第一个目标是开发一种用于端粒的单杂交系统 结合蛋白是在上一个资助期开发的。其中之一 杂交试验将用于鉴定新的端粒结合蛋白和 以确定已知蛋白质的结合要求。第二 目标是继续分析cdc13p，这是一种在最后一段中显示的蛋白质 体外结合单链TG1-3端粒DNA的资金期 以及体内的端粒。工作模式是物理存在 Cdc13p限制细胞周期调控的C链降解 端粒DNA和招募端粒酶到端粒。为了测试这一点 假说、生化和遗传方法将被用来确定 如果cdc13p与Pollp和estlp的相互作用，如两个 混合实验，调节C-链降解或端粒酶 复制。将确定cdc13p的磷酸化位点并 然后突变以评估cdc13p磷酸化的重要性。 功能。染色质免疫沉淀(CHIP)将用于 确定cdc13p是否与被认为是 存在于整个细胞周期和/或长的瞬变尾巴 目前处于S阶段的末期。第三个目标是确定 染色质组装因子I的大亚基(CAF-1)可降低 端粒(端粒)附近转录抑制的稳定性 位置效应，TPE)。将使用芯片和遗传方法来 确定组蛋白乙酰化对CAF-1介导的作用是否重要 TPE和IF-SIR蛋白与端粒或亚基相关的遗传 在缺少CAF-1的细胞中，端粒DNA会发生改变。第四个目标是 确定其转录状态是否已知的单个端粒 定位于核的一个特定的亚室。会是 确定本地化是否受转录状态的影响 端粒或通过减少TPE的突变。有越来越多的 推测端粒同时影响衰老和肿瘤的发生 人类。从酵母到人类，端粒都保存得很好。一项分析 影响端粒结构或功能的酵母蛋白很可能 与理解人类的遗传不稳定性有关。

Telomeres, the ends of eukaryotic chromosomes, are essential for the complete replication and stable maintenance of Saccharomyces chromosomes. Telomeres protect chromosomes from degradation, help the cell distinguish intact from broken DNA, provide a substrate for telomerase, and affect the transcription of nearby genes. The overall goal of this project is to identify structural proteins that interact with telomeres and to determine how they contribute to telomere function. The first goal is to exploit a one hybrid system for telomere binding proteins that was developed in the last funding period. The one hybrid assay will be used to identify new telomere binding proteins and to determine the binding requirements of known proteins. The second goal is to continue analysis of Cdc13p, a protein shown in the last funding period to bind single strand (ss) TG1-3 telomeric DNA in vitro and telomeres in vivo. The working model is that the physical presence of Cdc13p limits the cell cycle regulated degradation of C-strand telomeric DNA and recruits telomerase to the telomere. To test this hypothesis, biochemical and genetic approaches will be used to determine if the interaction of Cdc13p with Pollp and Estlp, as detected by two hybrid experiments, regulates either C-strand degradation or telomerase replication. The sites of Cdc13p phosphorylation will be determined and then mutated to assess the importance of phosphorylation to Cdc13p function. Chromatin immuno-precipitation (ChIP) will be used to determine if Cdc13p associates with the short ss tails thought to be present throughout the cell cycle and/or the long transient tails present at the end of S phase. The third aim is to determine how the large subunit of chromatin assembly factor I (CAF-1) reduces the stability of transcriptional repression near telomeres (telomere position effect, TPE). ChIP and genetic approaches will be used to determine if histone acetylation is important for CAF-1 mediated inheritance of TPE and if Sir protein association with telomeric or sub- telomeric DNA is altered in cells lacking CAF-1. The fourth aim is to determine if an individual telomere whose transcriptional state is known is localized to a specific sub-compartment of the nucleus. It will be determined if localization is affected by the transcriptional state of the telomere or by mutations that reduce TPE. There is increasing speculation that telomeres affect both aging and tumorigenesis in humans. Telomeres are well conserved from yeast to humans. An analysis of yeast proteins that affect telomere structure or function is likely to be relevant to an understanding of genetic instability in humans.