Real-time imaging of rotation during synthesis by the replisome

Abstract 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. Competing Interest Statement The authors have declared no competing interest. Footnotes We have added the results from further experiments, analysis, and revised the text and figures accordingly. Experiments with pre-assembly conditions have been added as well as the addition of gyrase. In addition to revisions of main text figures, we have added 8 additional supplemental figures and revised Table S1 to include the results from new experiments.