STRUCTURE & BEHAVIOR OF YEAST TELOMERES

端粒是真核生物染色体的末端， 酵母菌的完全复制和稳定维持 染色体 端粒保护染色体不被降解， 细胞区分完整的和断裂的DNA， 端粒酶，并影响附近基因的转录。 整体 这个项目的目标是确定结构蛋白， 并确定它们如何对端粒起作用 功能 第一个目标是开发一个端粒的单杂交系统 在上一个资助期开发的结合蛋白。 所述一 杂交分析将用于鉴定新端粒结合蛋白， 以确定已知蛋白质的结合要求。 第二 我们的目标是继续分析Cdc 13 p，这是一种在最后一次实验中显示的蛋白质。 体外结合单链（ss）TG 1 -3端粒DNA的资助期 和端粒。 工作模型是， Cdc 13 p限制了细胞周期调控的C链降解 端粒DNA和招募端粒酶端粒。 为了验证这一 假设，生物化学和遗传学方法将用于确定 如果Cdc 13 p与Pollp和Estlp的相互作用，如通过两个 杂交实验，调节C链降解或端粒酶 复制的将确定Cdc 13 p磷酸化的位点， 然后突变以评估磷酸化对Cdc 13 p的重要性， 功能 染色质免疫沉淀（ChIP）将用于 确定Cdc 13 p是否与被认为是 存在于整个细胞周期和/或长的瞬时尾 在S期结束时出现。 第三个目标是确定 染色质组装因子I大亚基（CAF-1）降低了 端粒附近转录抑制稳定性 位置效应，TPE）。 ChIP和遗传方法将用于 确定组蛋白乙酰化是否对CAF-1介导的 TPE遗传和if Sir蛋白与端粒或亚端粒相关 端粒DNA在缺乏CAF-1的细胞中改变。 第四个目标是 确定转录状态已知的单个端粒 位于细胞核的特定亚区室。 将 确定定位是否受转录状态的影响， 端粒或减少TPE的突变。 人们日益 推测端粒影响衰老和肿瘤发生， 人类 从酵母到人类，端粒都很保守。 分析 影响端粒结构或功能的酵母蛋白质 与人类遗传不稳定性的理解相关。

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.