Torsional dynamics of replication and transcription revealed using a toolbox of fluorescent DNA topology sensors

• Applicant: Professor Dr. Karl Duderstadt
• Subject Area: Biophysics
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517791312

The most essential task for the preservation and proliferation of all cellular life on earth is the faithful retrieval and duplication of genomic information. Cells rely on DNA for storage of this vital information, which offers many advantages including sequestration of nucleotides to reduce damage and two-fold redundancy to facilitate repair. Nevertheless, the double-helical structure of DNA poses challenges when the information-rich DNA bases must be accessed during transcription and DNA replication. Separation of the two strands leads to overtwisting and the formation of positive supercoils that must be resolved quickly to avoid extreme torques that can disrupt essential cellular processes, lead to irreversible chromosome damage, and ultimately cell death. Despite the critical importance of these events for cellular life, we have very limited knowledge about the DNA topology dynamics that occur when genomic information is accessed due to few methods suitable for real-time studies. Therefore, we propose the development of a toolbox of fluorescent DNA topology sensors that will enable real-time studies of DNA topology dynamics. To identify and quantitatively characterize fluorescent DNA topology sensors we will develop a screening platform using a calibrated collection of topological rulers. Ensemble gel-based approaches combined with the screening platform will identify fluorophores and fluorescently labeled factors with unique DNA topology recognition properties. In parallel, building on our past successes, we will develop single-molecule fluorescence imaging approaches to study transcription and DNA replication during encounters with topological challenges in DNA. In the final stage of the project, these efforts will come together with fluorescent DNA topology sensors introduced to transcription and DNA replication assays. For the first time, these experiments will reveal the real-time dynamics of DNA topology during these essential cellular processes. These observations will clarify how torsional dynamics regulate transcriptional bursting and how topological energy is partitioned throughout the replication fork to avoid replication fork stalling and collapse. Taken together, the sensor screening platform we will develop and real-time observations we will perform will serve as a resource for all future efforts to quantitatively measure changes in DNA topology to address long-standing questions in chromosome biology and facilitate biotechnology applications.

• DFG Programme: Research Grants