The structure of Poz1-Tpz1 reveals a dimerization module in the fission yeast shelterin complex

Stutz, Cian. The structure of Poz1-Tpz1 reveals a dimerization module in the fission yeast shelterin complex. 2014, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_11106

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Linear eukaryotic chromosomes are characterized by having distinct DNA ends. These ends, designated telomeres, are threatened by continued shortening in replication, nucleolytic degradation and unwanted fusion by the DNA-repair machinery. In order to counteract these processes, eukaryotic cells have evolved a protective cap, designated shelterin, which is assembled at telomeric repeats. In fission yeast, shelterin consists of the Taz1-Rap1 front-end that binds double strand telomeric repeats and the Pot1-Tpz1-Ccq1 assembly that is associated with the single strand 3’ overhang. A small protein designated Poz1 bridges the two halves and has been shown to negatively regulate telomerase, the enzyme that synthesizes telomeric repeats. We set out to investigate the molecular details of the poorly characterized protein Poz1 and its interaction with Tpz1 in an effort to gain insight into the mechanisms of telomere length regulation.
Here we present the crystal structure of spPoz1_30-249 bound to spTpz1_475-508 at 2.4 Ǻ resolution. Our structure remarkably resembles the structure of the TRF-homology domain (TRFH) of the human shelterin components TRF1 and TRF2. TRFH acts as a dimerization module in human shelterin. We speculated that also Poz1 functions as a dimerization module in fission yeast shelterin, a hypothesis that we subsequently validated by mutational analysis and size-exclusion chromatography. We revealed that Poz1 by itself is monomeric and upon binding of Tpz1 a Poz1-Tpz1 heterotetramer is formed. Furthermore, we showed that also in vivo heterotetramerization is essential for maintaining proper telomere length. Together with the previously reported dimeric existence of Taz1 and Rap1, as well as the recent discovery that Pot1 can dimerize upon telomere binding, these findings suggest an overall dimeric arrangement of the components in the fission yeast shelterin complex.
We replaced Poz1 by the structurally similar human TRF2H-dimerization domain and its binding partner hApollo. While poz1Δ strains show very long telomeres, the TRF2H-Apollo strain showed slow progressive telomere shortening. We suggest that shelterin function was mostly restored because TRF2H-Apollo imitates the primary function of Poz1, which is linking the double-strand and the single-strand binding halves of shelterin complex in a dimeric fashion. Likely, slow telomere shortening is observed because the interactions between Rap1-Poz1-Tpz1 are likely dynamic and regulated, but TRF2H and Apollo were fused to Rap1 and Tpz1 respectively, not allowing association and dissociation, leading to disrupted telomere regulation.
Furthermore, the structure revealed a zinc binding site at the binding interface of Poz1 and Tpz1. Disruption of the zinc site does not affect Poz1-Tpz1 interaction, but leads to decreased solubility of the complex in vitro and long telomeres in vivo. Consequently, binding of zinc is essential for the structural integrity of the complex.
Ultimately, by determining the crystal structure of Poz1_30-249+Tpz1_475-508, we uncovered new details of the architecture of the fission yeast shelterin complex and revealed striking similarities to structural elements found in human shelterin. Furthermore we examined the delicate interplay between the shelterin components and how essential its integrity is for proper telomere length homeostasis.
Advisors:Gasser, Susan
Committee Members:Baumann, Peter
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI > Quantitative Biology > Nuclear organization in development and genome stability (Gasser)
UniBasel Contributors:Gasser, Susan
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:11106
Thesis status:Complete
Number of Pages:128 p.
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:52
Deposited On:02 Feb 2015 13:20

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