Regulating telomere structure and telomerase recruitment

已有研究表明，纤毛虫的大核端粒在体内采用反平行的G-四链结构，并通过端粒末端结合蛋白(TEBP)的一个亚单位与亚核结构结合，可能是核基质或支架。在复制过程中，端粒之间的相互作用以及G-四链结构都得到了解决。这项建议的一个范围是分析G-四链DNA结构在这些细胞中如何在复制过程中及时分解，确定亚核结构中的TEBPot相互作用，并分析这种相互作用在复制过程中是如何分解的。为此，我们将分析在复制过程中TEBPü的磷酸化状态，以及这如何有助于在体外和体内解析G-四链结构。将确定TEBpa在复制过程中的命运，并测试端粒酶在G-四链结构拆分中的可能贡献。通过改进的Western分析或染色质免疫沉淀可以确定TEBPA与端粒的相互作用伙伴，并将在体外和体内测试其在S阶段可能的修饰。虽然一般认为哺乳动物体内的端粒采用T-环结构，但越来越多的间接证据表明，它们也可能采用G-四链结构。使用在纤毛虫中成功应用的相同方法，我们将尝试测试哺乳动物端粒是否在体内采用G-四链结构，以及哺乳动物与纤毛虫TEBP的同源物如何形成这种结构。这是这项提议的第二个范围，如果成功，这将开辟一个全新的研究领域。

It has been well demonstrated that the macronuclear telomeres of stichotrichous ciliates adopt the antiparallel G-quadruplex structure in vivo and adopt this structure while bound via the asubunit of the telomere end-binding protein (TEBP) to a subnuclear structure, possibly the nuclear matrix or scaffold. In the course of replication, both, the interaction of telomeres as well as G-quadruplex structure become resolved. One scope of this proposal is to analyze how G-quadruplex DNA structure becomes resolved during replication in a timely manner in these cells, to identify the TEBPot interaction panner in the subnuclear structure and to analyze how this interaction is resolved during replication. For this we will analyze the phosphorylation status of TEBPß during replication and how this contributes to the resolution of G-quadruplex structure in vitro and in vivo. The fate of TEBPa during replication will be determined and a possible contribution of telomerase in the resolution of G-quadruplex structure tested. The interaction partner to which TEBPa tethers telomeres can be identified either by a modified Western analysis or chromatin immunoprecipttation and its possible modification during S-phase will be tested in vitro and in vivo. Although it is generally assumed that mammalian telomeres adopt the T-loop structure m vivo there is increasing indirect evidence that they may also adopt G-quadruplex structure. Using the same approach as successfully applied in ciliates we will make an attempt to test whether mammalian telomeres adopt G-quadruplex structures in vivo and how this structure may be formed by mammalian homologs to the ciliate TEBP. This is the second scope of this proposal and, if successful, this would open up a whole novel area of research.