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

我们之前已经表明，纤毛虫大核纳米染色体的端粒在体内采用G-四链体结构，其形成由两个端粒末端结合蛋白TEBPa和TEBPb控制，磷酸化的TEBPb参与S期的端粒酶募集，TEBPb的磷酸化以及端粒酶结合，是复制过程中端粒G-四链体结构解旋的必要先决条件。最近，我们发现了一个RecQ样解旋酶，它在Sphase期间与端粒酶相关，被这种酶招募到端粒，并在复制过程中参与G-四链体解旋。在这项建议中，我们将重新定义TEBPb的端粒酶的招聘的结构要求，并将其与最近发表的关于端粒酶的招聘的人类TEBPb同源物TPP 1的数据进行比较。为此，我们将表达TEBPb，如果可能的话，端粒酶，构建各种缺失突变体，并通过微量热泳和免疫共沉淀实验测试它们的相互作用。我们将进一步研究RecQ样解旋酶在体外的活性，并试图了解它与端粒酶的关系以及这种复合物如何被招募到端粒的机制。虽然我们已经表明，有些实验可以立即完成，但我们意识到，有些方法并不是微不足道的，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.