Engineering yeast chromosomal telomeres with a bacteriophage system

Area of Science:

• Molecular Biology
• Genetics
• Synthetic Biology

Background:

• Eukaryotes possess linear chromosomes protected by conserved telomere-telomerase systems, evolved over a billion years.
• These systems are crucial for protecting chromosomal ends and regulating physiological functions.

Purpose of the Study:

• To replace the endogenous eukaryotic telomeres in Saccharomyces cerevisiae with the prokaryotic TelN/tos system from bacteriophage N15.
• To investigate the functionality and implications of this telomere system replacement in a eukaryotic host.
• To develop a novel platform for large-scale DNA manipulation.

Main Methods:

• Interruption of the MRX/Sae2 pathway in yeast to facilitate telomere replacement.
• Introduction of the prokaryotic TelN/tos system to serve as a functional telomere.
• Adaptive evolution of engineered yeast strains to identify beneficial mutations (TEL1, CYR1).
• Development of a tos-YAC (Yeast Artificial Chromosome) system for DNA assembly.

Main Results:

• The prokaryotic telomeres successfully protected linear chromosomal ends and prevented genetic instability in yeast.
• Adaptive evolution identified mutations restoring MRX/Sae2 activity, enhancing yeast fitness and meiotic capacity.
• Engineered telomeres localized deeper into chromosomes and showed increased interactions with adjacent regions.
• The tos-YAC system enabled stable maintenance and iterative assembly of large DNA fragments (1.23–2.77 Mb).

Conclusions:

• Functional equivalence between divergent eukaryotic and prokaryotic telomere systems was demonstrated.
• The findings suggest potential natural origins for such system divergence, possibly via horizontal gene transfer.
• The engineered strains and tos-YAC system provide a robust platform for large-scale DNA manipulation and synthetic biology applications.