Controling telomere replication - telomerase recruitment and the unwinding of telomeric G-quadruplex structure

我们已经证明，纤毛虫大核纳米染色体的端粒在体内采用g -四重体结构，其形成受两种端粒末端结合蛋白TEBPa和TEBPb的控制，TEBPb的磷酸化参与了s期端粒酶的募集，TEBPb的磷酸化和端粒酶的结合是端粒g -四重体结构在复制过程中解绕的必要前提。最近，我们发现了一种类似recq的解旋酶，它在酶相期间与端粒酶相关，通过这种酶被募集到端粒，并在复制过程中参与g -四重体解绕。在本文中，我们将描述TEBPb对端粒酶募集的结构要求，并将其与最近发表的关于人类TEBPb同源物TPP1募集端粒酶的数据进行比较。为此，我们将表达TEBPb和端粒酶（如果可能的话），构建各种缺失突变体，并通过微热电泳和共免疫沉淀实验检测它们的相互作用。我们将在体外进一步表征recq样解旋酶的活性，并试图了解其与端粒酶的关联以及该复合物如何被募集到端粒的机制。虽然我们已经表明，有些实验可以立即进行，但我们也意识到，有些方法不是微不足道的，有时需要冒险的实验，我们依赖于合作伙伴的帮助。

We have shown before that the telomeres of macronuclear nanochromosomes in ciliates adopt the G-quadruplex structure in vivo, that its formation is controlled by two telomere-end binding proteins, TEBPa and TEBPb, that phosphorylated TEBPb is involved in telomerase recruitment during S-phase and that both, phosphorylation of TEBPb as well as telomerase binding, are necessary prerequisites for the unwinding of telomeric G-quadruplex structure during replication. Very recently, we identified a RecQ-like helicase which becomes associated with telomerase during Sphase, recruited to telomeres by this enzyme and is involved in G-quadruplex unwinding during replication. In this proposal we will characterise the structural requirements of TEBPb for the recruitment of telomerase and compare it to recently published data on the recruitment of telomerase by the human TEBPb homolog TPP1. For this we will express TEBPb and, if possible, telomerase, construct various deletion mutants and test their interaction by microscale thermophoresis and coimmunoprecipitation experiments. We will further characterise the activity of the RecQ-like helicase in vitro and try to understand its association with telomerase and the mechanism how this complex becomes recruited to the telomeres. While we al-ready have shown that some of the experiments can be done immediately we are aware that some approaches are not trivial to do and will require sometimes risky experiments for which we rely on the help of co-operation partners.