From in vitro screens to telomeric phenotypes: identification and initial characterisation of two novel telomere-associated proteins in saccharomyces cerevisiae

Abstract

A telomere (literally, “end piece” from Greek télos “end” and méros “part”) is a specialised nucleoprotein structure located at each end of a linear eukaryotic chromosome. Its presence is essential for genome stability and the integrity of DNA termini, ensuring their intactness by preventing gratuitous fusion and recombination events from occurring. In addition, telomeres, together with the enzyme telomerase, facilitate the replication and maintenance of chromosomal ends, thereby preventing premature onset of senescence and the progressive loss of genetic material. In most organisms, telomeric DNA consists of short, repetitive sequences. The nominally canonical TTAGGG repeat is believed to be ancient and broadly conserved among eukaryotes. It is all the more remarkable, then, that telomeric repeat sequences in Saccharomycotina yeasts are exceptionally diverse – typically TG-degenerated, generally not G/C-rich, and varying in both the overall length and tandem repeat sequence patterns among species. One of the most widely used model organisms, the budding yeast Saccharomyces cerevisiae, is evolutionarily young on the yeast phylogenetic tree. Interestingly, species from more basal clades tend to retain greater similarity to the canonical TTAGGG repeat, whereas more derived species show increasing divergence. Consequently, telomere-binding and telomere-associated proteins (TBPs) in yeasts are not necessarily conserved and may vary across clades, occasionally even swapping their functional roles. In this thesis, species-specific telomeric repeat sequences were used in in vitro pull-down assays to investigate the co-evolution of TBPs within the Ascomycota group of yeasts, with the aim of identifying novel telomere binders. As a result, two TBPs – transcription factors and paralogs, Tda9 and Rsf2 – were identified as telomere-binding proteins in several yeast species in vitro, and also confirmed to bind telomeric DNA in vivo in S. cerevisiae. Subsequent experiments focused on S. cerevisiae, revealing that deletion of these proteins impacts several telomeric features. These include changes in telomere length and telomeric silencing, impaired formation of Type II survivors during replicative senescence and telomere lengthening upon overexpression of one of the two proteins. Overall, this study aims to contribute to the broader understanding of telomere integrity in yeast and, more generally, to expand our knowledge of telomere biology, composition and function. In doing so, it offers an incremental yet meaningful perspective that could, in time, support advances in therapeutic strategies related to cancer and healthy ageing.