Retzer, T. M. | Search bioRxiv

bioRxiv2025

Real-time imaging of rotation during synthesis by the replisome

During chromosome replication, unwinding by the helicase and synthesis by the polymerases can lead to overwinding and supercoiling of DNA. The mechanical consequences of these events and resulting local dynamics at the replication fork are not well understood. To address these issues, we developed a transverse DNA flow-stretching approach to spatially resolve the parental, leading and lagging strands in real-time. Using bacteriophage T7 as a model system, this approach revealed bursts of high-speed replisome rotation that support continuous DNA synthesis. Surprisingly, excessive rotation does not reduce replisome speed, but increases pausing, reduces processivity, and increases the number of polymerases. Taken together, our observations reveal intrinsic pathways to overcome challenges posed by unfavorable DNA topologies during DNA replication.

biophysics↗

bioRxiv2021

Mars, a molecule archive suite for reproducible analysis and reporting of single molecule properties from bioimages

The rapid development of new imaging approaches is generating larger and more complex datasets revealing the time evolution of individual cells and biomolecules. Single-molecule techniques, in particular, provide access to rare intermediates in complex, multistage molecular pathways. However, few standards exist for processing these information-rich datasets, posing challenges for wider dissemination. Here, we present Mars, an open-source platform for storing and processing image-derived properties of biomolecules. Mars provides Fiji/ImageJ2 commands written in Java for common single-molecule analysis tasks using a Molecule Archive architecture that is easily adapted to complex, multistep analysis workflows. Three diverse workflows involving molecule tracking, multichannel fluorescence imaging, and force spectroscopy, demonstrate the range of analysis applications. A comprehensive graphical user interface written in JavaFX enhances biomolecule feature exploration by providing charting, tagging, region highlighting, scriptable dashboards, and interactive image views. The interoperability of ImageJ2 ensures Molecule Archives can easily be opened in multiple environments, including those written in Python using PyImageJ, for interactive scripting and visualization. Mars provides a flexible solution for reproducible analysis of image-derived properties, facilitating the discovery and quantitative classification of new biological phenomena with an open data format accessible to everyone.

biophysics↗