Telomere architecture and maintenance in schizosaccharomyces pombe

Abstract

Telomeres are specialized nucleoprotein structures located at the ends of eukaryotic chromosomes that promote genomic integrity. Impaired telomere function has been implicated in cancer as well as several degenerative diseases such as dyskeratosis congenita. Telomeric DNA consists of tandem G-rich repeats with a single-stranded 3' overhang that can be extended by telomerase, a reverse transcriptase, which counteracts the shortening caused by incomplete replication or nucleolytic degradation at chromosome ends. Telomere binding proteins, specifically recruited by the telomeric DNA, provide essential functions for the protection and maintenance of telomeres. The telomere maintenance machinery of fission yeast Schizosaccharomyces pombe, a genetically tractable organism, shares fundamental similarities with that of humans, making it an ideal model system to study telomere biology. In fission yeast, the double-stranded telomeric repeats are directly bound by Taz1, which recruits Rap1. Pot1, aided by Tpz1, binds the single-stranded DNA and Poz1 forms a bridge between Taz1/Rap1 and Pot1/Tpz1. Different parts of this telomeric complex contribute differently to telomere length equilibrium. Deletion of Taz1, Rap1 or Poz1 results in substantial telomere elongation suggesting their negative roles in length regulation. In contrast, deletion of Tpz1 or Pot1 causes rapid telomere loss, suggesting that they play positive roles in telomere protection and telomerase recruitment and activation. The molecular details of the interactions between these proteins and their contributions to the maintenance of telomere length equilibrium is not well understood. By systematically dissecting the role of each component in the complex, we demonstrate that each individual interaction within the Taz1-Rap1-Poz1-Tpz1 unit is critical for length regulation but can be replaced with a static linker, arguing against a necessity for dynamic regulation of the interactions. Furthermore, Rap1 and Poz1 function as interaction modules to provide a molecular bridge between Taz1 and Tpz1 which can be replaced by a short linker. Our results suggest that the architecture of the telomere complex, rather than the protein components per se, is critical for the maintenance of telomere length equilibrium. We established minimal complexes (mini-telosomes) for telomere length regulation. Further characterization of the mini-telosomes provides new insights into the separation of different telomere functions. In addition, we investigated the molecular details of the Poz1-Tpz1 interaction, which is at the interface of negative and positive regulators. The crystal structure was solved by the Thomä lab and together with my in vivo work demonstrates a conserved binding motif used at different parts of telomeric complexes in human and S. pombe. Furthermore, a zinc ion is bound at the interface of the Poz1-Tpz1 interaction and promotes the interaction as well as telomere length regulation. In addition, Poz1-Tpz1 forms a heterotetrameric arrangement, which may provide the basis for higher order structures.