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. 

Methods
This dataset was collected using Micromanager 2.0. Videos are sequences of tiff images. They are most easily opened using FIJI with the Mars update site activated. They can then be opened just using the open command in Fiji and selecting one of the images or the metadata.txt file