Engineering yeast chromosomal telomeres with a bacteriophage system

All eukaryotes, including yeast, plants, animals and humans, possess linear chromosomes. The conserved eukaryotic telomere-telomerase systems, originated and evolved over 1 billion years, protect the chromosomal ends and regulate critical physiological functions through complex networks. In this study, we replace the endogenous eukaryotic telomeres in the single-chromosome yeast Saccharomyces cerevisiae with the prokaryotic telomere system TelN/tos from the Escherichia phage N15, which forms a closed hairpin structure, by interrupting the MRX/Sae2 pathway. The prokaryotic telomeres effectively protect linear chromosomal ends and prevent genetic instability. Through adaptive evolution, we identify yeast strains harboring additional mutations (TEL1 and CYR1) that restore functional MRX/Sae2 activity, thereby improving host fitness and meiotic capacity. Interestingly, the two-associated TelN/tos telomeres position deeper into chromosomes and exhibit increased interactions with their adjacent regions. The successful replacement of a complex eukaryotic chromosomal telomere with a simple bacteriophage system demonstrates functional equivalence between these divergent systems, implying possible natural origins of such stochasticity (e.g., horizontal gene transfers). Furthermore, these engineered strains facilitate development of a tos-YAC system that enabled iterative assembly and stable maintenance of megabase-level heterogeneous DNA (1.23-2.77 Mb), providing a robust platform for large-scale DNA manipulation.