Geometry of braided DNA dictates supercoiling partitioning

DNA's helical structure requires the replisome to rotate relative to the parental DNA during replication, generating supercoiling that partitions ahead and behind the fork. The influence of DNA substrate geometry on supercoiling partitioning and torsional resistance remains unclear. Here, we have engineered DNA-braiding substrates with end separations found during replication, measured braiding torques using an angular optical trap, and interpreted the results using Monte Carlo simulations. A substrate with small separations readily initiates braiding, suggesting that the fork would rotate during replication, partitioning supercoiling behind the replisome and reducing torsional resistance. In contrast, a substrate with a substantial separation at one end imposes a pronounced 'torsional barrier' that hinders braiding initiation, suggesting a lack of fork rotation during replication, so supercoiling would partition ahead of the replisome. Our findings reveal a physical mechanism in which DNA fork geometry could modulate replisome dynamics, manage topological stress, and regulate replication progression in vivo.