Structure and function of factors mediating Tetrahymena telomerase action at telomeres

• Applicant: Dr. Christina Helmling
• Subject Area: Structural Biology ; Biological and Biomimetic Chemistry
• Term: from 2017 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 390533466

The ends of linear chromosomes in eukaryotic organisms consist of DNA-protein complexes termed telomeres. Telomere proteins bind specifically to double- and single-stranded G-rich repeats of telomeric DNA to protect these ends from recombination and degradation. The inability of the cellular DNA replication machinery to fully replicate telomeric DNA leads to gradual telomere shortening, which is rescued, in part, by the specialized reverse transcriptase telomerase. Telomerase activity is directly coupled to telomere length maintenance, which regulates the normal life span of cells. Telomerase has emerged as a major target for therapeutic intervention due to the aberrant upregulation of telomerase in tumor cells, which is responsible for their immortal phenotype. The overall architecture and subunit arrangement of the telomerase holoenzyme from the ciliate Tetrahymena was recently solved by electron microscopy studies in the host laboratory of Juli Feigon. The results of these studies propose a difference in the regulatory mechanism underlying telomere self-renewal in Tetrahymena compared to humans. While in humans, recruitment of telomerase to its cellular substrate is mediated by a subunit of the telomere (TPP1), the proposed telomere counterpart in Tetrahymena (Tpt1) appears to function only as a protector of telomeres, while the recruitment role of TPP1 is assigned to an integral part of the telomerase holoenzyme (p50). In this arrangement of subunits, either Tpt1 in complex with the single-stranded telomere binding protein Pot1 occupies and protects telomeric repeats, or telomerase binds and catalyzes repeat synthesis. Alternate binding of these two multisubunit complexes is responsible for telomere length regulation.The overall objective of the project presented here is to investigate structure and function of the largely uncharacterized telomerase inhibitor complex Tpt1-Pot1a in comparison to the ‘activating’ subunits within the telomerase holoenzyme (p50-TEB). To achieve this goal, first structure and dynamics of Tpt1 in isolation and in complex with Pot1a will be investigated using NMR-spectroscopy and X-ray crystallography as complementary methods. Structural data can be directly compared to ongoing work on the telomerase subunits in the host laboratory, which is directed towards improving the structure of these competing counterparts. In addition, biochemical studies will address ssDNA binding properties of Tpt1-Pot1a together with telomerase activity assays in direct competition for substrate DNA binding. The insights derived from the project proposed here should contribute significantly to improve understanding regarding differences in structure and telomeric DNA binding properties of Tpt1-Pot1a and telomerase that ultimately coordinate telomere length maintenance.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Juli Feigon