STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

描述(申请人提供)：这项资助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。在包括酿酒酵母在内的大多数真核生物中，端粒酶是由一种特殊的逆转录酶合成的，在酵母中，端粒酶的作用既是细胞周期调节的，发生在S晚期，也是长度调节的，优先发生在较短的端粒上。核心端粒酶由TLC1基因编码的端粒酶RNA和EST2编码的催化逆转录酶组成。与酵母染色体末端的单链TG1-3尾巴结合的CdCl3p和Estlp和Est3p一样，对体内的四聚酶作用也是必不可少的，这两种蛋白质的功能尚不清楚。此外，ATM样激酶Tel1p和MREL 1-Rad50-Xrs2(MRX)复合体在体内促进端粒酶作用的途径是相同的。在上一个资助期，我们建立了一种灵敏的染色质免疫沉淀分析方法，可以检测端粒上的est2p、est1p和CDC13p。本实验表明，cdcl3p在整个细胞周期中都是四聚体结合的，但在端粒酶作用的S晚期，其结合量大大增加，当端粒酶激活时，est2p不仅在S晚期结合端粒，当端粒酶不激活时，它也结合在G1期和S早期。ESTLP结合仅限于S晚期。这些数据表明了一种模型，其中CDC 13p多聚体和ESTT p结合激活了原本不活跃的端粒结合的EST2plTLC t复合体。这项拨款建议进行实验，以测试和扩展这一模型。Est LP的作用将通过体内和体外试验进一步确定。我们将确定Ku复合体是否在GT的端粒上含有Est2p/TLct，以及端粒的封顶功能是否需要它的存在。我们将确定est3p和MRX是否与端粒结合，这种结合是否对它们促进端粒酶的作用重要，以及它们的缺失是否影响其他端粒酶成分的结合。我们将确定短端粒的优先延长是否由ESTLP和/或MRX介导。最后一个目的描述了细胞学方法，以确定端粒定位到核周是否对转录沉默或端粒的端粒酶延长很重要。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，以及作用于四聚体的蛋白质，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理解人类的遗传不稳定性有关。

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.