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

我们以前已经证明纤毛虫大核纳米染色体的端粒在体内采用G-四链结构，它的形成受两个端粒末端结合蛋白TEBPA和TEBPB的控制，磷酸化的TEBPB参与了S期的端粒酶募集，并且两者都是复制过程中解开端粒G-四链结构的必要条件。最近，我们发现了一种类似RecQ的解旋酶，它在S期与端粒酶相关，被该酶招募到端粒，并参与复制过程中G-四链的解离。在这项建议中，我们将表征TEBPB对端粒酶招募的结构要求，并将其与最近发表的人类TEBPB同源物TPP1招募端粒酶的数据进行比较。为此，我们将表达TEBPB，并在可能的情况下表达端粒酶，构建各种缺失突变体，并通过微型热电泳法和免疫共沉淀实验测试它们之间的相互作用。我们将在体外进一步鉴定RecQ样解旋酶的活性，并试图了解它与端粒酶的关联以及这种复合体如何被招募到端粒的机制。虽然我们已经证明，一些实验可以立即完成，但我们意识到，有些方法不是轻而易举的，有时需要进行危险的实验，而我们需要依靠合作伙伴的帮助。

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.