Optimisation of the Schizosaccharomyces pombe urg1 expression system

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Studying protein function in vivo requires precise control over protein expression levels.
• Existing transcriptional control systems in fission yeast face limitations in induction speed and achieving low basal transcription in the OFF-state.

Purpose of the Study:

• To develop versatile modules for fine-tuning the Purg1 promoter's OFF-levels in fission yeast.
• To maintain a wide dynamic range of gene expression control.
• To integrate Purg1 control with auxin degron technology for null-like phenotypes.

Main Methods:

• Cre recombination-mediated gene regulation using the Purg1 promoter.
• Development of modules to reduce basal transcription.
• Integration of Purg1 transcriptional control with auxin degron system.
• Application in studying DNA double-strand break formation, replication fork arrest, and post-replication repair.

Main Results:

• Achieved significant reduction in Purg1 OFF-levels while maintaining a dynamic range of approximately 75-fold.
• Demonstrated improved regulation of HO-dependent DSB formation.
• Showcased enhanced control over Rtf1-dependent replication fork arrest.
• Successfully regulated Rhp18(Rad18)-dependent post-replication repair.

Conclusions:

• The developed modules offer enhanced, tunable transcriptional control in fission yeast.
• This system facilitates the study of essential gene functions and complex cellular processes.
• The tools enable precise manipulation of protein levels, aiding in the understanding of DNA repair mechanisms.