Struktur und Funktion von Telomerase-Telomer Vermittlungsfaktoren
Biologische und Biomimetische Chemie
Zusammenfassung der Projektergebnisse
The ends of linear chromosomes (telomeres) progressively shorten with each cell cycle leading to senescence, apoptosis and genome instability. Telomeres are maintained by the specialized reverse transcriptase telomerase. Telomeric repeat DNA is protected by single-stranded and double-stranded DNA binding proteins that both recruit and regulate telomerase action at telomeres. We investigated structure and function of the Tetrahymena telomeric DNA binding heterodimeric protein complex Tpt1-Pot1a as well as its interaction with telomerase to advance the overall understanding of telomere length regulation. We solved the NMR structure of Tpt1OBN, a potentially key domain in recruitment and regulation of telomerase access to telomeres, and revealed profound differences in global fold compared to functional homologues. Tpt1 does not contain the typical ‘TEL-patch’ residues that were found to interact with telomerase in human TPP1 due to a relocation in α2 and L3-4 being significantly shortened. The domain adopts an usual β-barrel fold with six antiparallel β-strands that introduces an additional a large flexible loop L5-6. L5-6 represents and interesting potential new docking interface for telomerase. Alternatively, an interaction between telomerase and Tpt1 could lead to a structural rearrangement within Tpt1. NMR backbone assignments of Pot1aOB3 reveal that the domain is structurally very similar to the predicted homology model. We found the Pot1aOB3-Tpt1OBN-PBD complex to crystallize in space group P21 with 1.9 Å resolution and hope to obtain further information on the structural relation between the Pot1a-Tpt1 complex and the available homology structure for human TPP1-POT1 in the near future. Electrophoretic mobility shift assays performed with Pot1aFL/Tpt1OBN-PBD and (GTTGGG)3/(GTTGGG)5 show that Pot1aFL specifically binds to telomeric DNA with ~100 nM affinity, while the addition of Tpt1 enhances Pot1a binding affinity ~2.5-fold. The Tetrahymena telomerase protein Teb1 binds specificlly to telomeric DNA with 5 nM affinity suggesting a potential displacement of Pot1a from telomeres in the presence of high concentrations of telomerase. However, addition of a large excess of Pot1a (> 2000x) during direct telomerase activity assays was found to completely inhibit telomerase activity. Telomerase co-assembled with Tpt1 shows a different activity profile compared to telomerase only. Further, Tpt1 was found to partially rescue the inhibitory effect of Pot1a in telomerase activity assays by functioning as a potential bridge between Pot1a and telomerase. Pot1a only assembles with telomerase in the presence of DNA containing at least five consecutive telomeric repeats. This result suggests that telomerase does not directly interact with telomerase via Tpt1 but independently binds to extending telomeric DNA. Telomerase activity assays performed in the presence of excess of Tpt1 (1 µM) shows a telomerase processivity enhancement of 4-5 fold and telomerase activity enhancement by ~2-fold induced by Tpt1. In the presence of the Tpt1-Pot1a complex, telomerase processivity is enhanced 5-fold while the overall activity is significantly reduced. Therefore, Pot1a does not influence telomere extension by telomerase once telomerase has gained access to telomeres but reduces activity by sequestering free substrate DNA. Biochemical studies of the Tpt1-Pot1a complex have revealed significant functional similarities between human TPP1 and Tetrahymena Tpt1. However, from a structural perspective, we also identified profound differences between the Tetrahymena Tpt1 and human TPP1, in particular with respect to the telomerase interaction interface. The results obtained during the funded project provide the basis for detailed mechanistic studies of telomere length regulation.
