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

Fachliche Zuordnung Allgemeine Genetik und funktionelle Genomforschung
Förderung Förderung von 2012 bis 2016
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 215322325
 
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.
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