STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

描述（由申请人提供）：该基金的长期目标是确定与酵母端粒相互作用的蛋白质如何影响端粒功能。 在大多数真核生物中，包括酿酒酵母，端粒DNA是由一种专门的逆转录酶端粒酶合成的。在酵母中，端粒酶作用既受细胞周期调节，发生在S期晚期，又受长度调节，优先发生在短端粒。核心端粒酶由TLC 1基因编码的端粒酶RNA和EST 2编码的催化逆转录酶组成。与酵母染色体末端的单链TG 1 -3尾结合的Cdcl 3 p也是体内替托马酶作用所必需的，Estlp和Est 3 p是两种功能知之甚少的蛋白质。此外，Tel 1 p，ATM样激酶和Mrel 1-Rad 50-Xrs 2（MRX）复合物在相同的途径中起作用，以促进体内端粒酶作用。在上一个资助期内，我们开发了一种灵敏的染色质免疫沉淀试验，可以检测端粒上的Est 2 p、Est 1 p和Cdc 13 p。 Est 2 p不仅在端粒酶活性时的S期晚期与端粒结合，而且在端粒酶不活性时的G1期和S期早期也与端粒结合。Estlp结合仅限于S期晚期。这些数据表明了一种模型，其中Cdc 13 p多聚化和Esttp结合激活否则无活性的端粒结合的Est 2 plTLC t复合物。这项资助提出了实验来测试和扩展这个模型。Est lp的作用将使用体内和体外测定进一步定义。我们将确定Ku复合物是否在GT中的端粒处保持Est 2 p/TLCt，以及是否需要它的存在来实现端粒的封盖功能。我们将确定Est 3 p和MRX是否结合端粒，这种结合是否对它们在促进端粒酶中的作用很重要，以及它们的缺失是否影响其他端粒酶组分的结合。 我们将确定短端粒的优先延长是否是Estlp和/或MRX介导的。最后一个目标描述了细胞学方法，以确定是否端粒定位到核周边是重要的转录沉默或端粒酶延长端粒。越来越多的证据表明端粒复制对衰老和癌症都有影响。端粒功能，包括端粒沉默，以及作用于四粒的蛋白质从酵母到人类都是保守的。对影响酵母端粒的蛋白质的分析可能与对人类遗传不稳定性的理解有关。

DESCRIPTION (provided by applicant): The long-term goal of this grant is to determine how proteins that interact with yeast telomeres affect telomere function. In most eukaryotes, including Saccharomyces cerevisiae, telomeric DNA is synthesized by a specialized reverse transcriptase, telomerase, in yeast, telomerase action is both cell cycle regulated, occurring in late S phase, and length regulated, occurring preferentially at short telomeres. Core telomerase consists of telomerase RNA, encoded by the TLC1 gene, and the catalytic reverse transcriptase, encoded by EST2. Cdcl3p, which binds the single strand TG1-3 tails at the very ends of yeast chromosomes is also essential for tetomerase action in vivo as are Estlp and Est3p, two proteins whose functions are poorly understood. In addition, Tel1p, an ATM-like kinase and the Mrel 1-Rad50-Xrs2 (MRX) complex function in the same pathway to promote telomerase action in vivo. In the last funding period, we developed a sensitive chromatin immuno-precipitation assay that can detect Est2p, Est1p, and Cdc 13p at telomeres. This assay reveals that Cdcl3p is tetomere bound throughout the cell cycle but its binding increases greatly in late S phase, the time of telomerase action, Est2p binds telomeres not just in late S phase when telomerase is acti',,e but alsoin G1 and early S phase when it is not. Estlp binding is restricted to late S phase. These data suggest a model where Cdc 13p multimerization and Estt p binding activate an otherwise inactive, telomere bound Est2plTLC t complex. This grant proposes experiments to test and extend this model. The role of Est lp will be further defined, using both in vivo and in vitro assays. We wilt determine if the Ku complex holds Est2p/TLCt at the telomere in Gt and if its presence is needed for the capping function of telomeres. We will determine if Est3p and MRX bind telomeres, if this binding is important for their role in promoting telomerase, and if their absence affects binding of other telomerase components. We will determine if the preferential lengthening of short telomeres is Estlp and/or MRX mediated. The last aim describes cytological approaches to determine if telornere localization to the nuclear periphery is important for transcriptional silencing or for telomerase lengthening of telomeres. There is increasing evidence that telomere replication has effects on both aging and cancer. Telomere functions, including telomeric silencing, as well as the proteins that act at tetomeres are conserved from yeasts to humans. Analysis of proteins that affect yeast telomeres is likely to be relevant to an understanding of genetic instability in humans.