Torsion is a dynamic regulator of DNA replication stalling and reactivation.

Abstract

DNA's helical structure necessitates replisome rotation relative to DNA during replication, creating inevitable topological challenges. How replication generates and overcomes torsional stress remains unclear. Here, we developed a high-resolution, label-free, real-time assay to track DNA rotation by T7 replisome and its slowing under torsional stress. While helicase or DNA polymerase (DNAP) alone is a weak rotary motor, together they form the most powerful DNA rotary motor yet studied, generating ~22 pN·nm torque before stalling, twice that of E. coli RNA polymerase. Upon stalling, helicase-DNAP interactions stabilize the fork; without them, regression can extend hundreds of base pairs. Prolonged stalling inactivates the replisome, but excess DNAP, aided by interactions with helicase, promotes restart. Gyrase supports steady replication and enables timely restart of stalled forks. These findings demonstrate that helicase-DNAP synergy is essential for maintaining fork integrity under torsion, and that torsion is a key regulator of replication stalling and reactivation.