Regulating telomere structure and telomerase recruitment

研究表明，丝毛类纤毛虫的大核端粒在体内具有反平行的G-四链体结构，并通过端粒末端结合蛋白（TEBP）亚基与亚核结构（可能是核基质或支架）结合。在复制过程中，端粒的相互作用以及G-四链体结构都得到解决。该建议的一个范围是分析G-四链体DNA结构如何在这些细胞中及时地在复制过程中被解析，以鉴定亚核结构中的TEBPot相互作用panner，并分析这种相互作用如何在复制过程中被解析。为此，我们将分析TEBP在复制过程中的磷酸化状态，以及这如何有助于体外和体内G-四链体结构的解析。TEBPa在复制过程中的命运将被确定，并测试端粒酶在G-四链体结构解析中的可能贡献。TEBPa系链端粒的相互作用伴侣可以通过改良的Western分析或染色质免疫沉淀法鉴定，并且将在体外和体内测试其在S期期间的可能修饰。虽然一般认为哺乳动物端粒在体内采用T环结构，但越来越多的间接证据表明它们也可能采用G四链体结构。使用相同的方法，成功地应用于纤毛虫，我们将尝试测试哺乳动物端粒是否在体内采用G-四链体结构，以及如何形成这种结构可能是由哺乳动物同源的纤毛虫TEBP。这是该提案的第二个范围，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.