Abstract
During DNA replication, the replisome must rotate relative to the DNA substrate, generating supercoiling that must be partitioned in front of or behind the replisome. Supercoiling partitioned behind the replisome may intertwine (or braid) daughter DNA molecules and restrict chromosome segregation. Supercoiling partitioning and torsional resistance at the replisome should depend on the geometry of the two daughter DNA molecules, determined by their end separations. However, experimental investigation of DNA braiding under well-defined DNA geometry has proven challenging. Here, we present methods to engineer braiding substrates of defined geometry, from minimal to significant end separations. We then directly measured the torque required to braid these substrates using an angular optical trap (AOT) and found that the torque required to initiate the braiding during the first 0.5 turn critically depends on the end separation. Once braiding started, we found that the subsequent effective twist persistence length of DNA braiding is about 20-30 nm, insensitive to the end separations. Our work highlights the crucial role of braiding geometry in dictating supercoiling partitioning and torque build-up during replication. It suggests that dynamic modulation of end separation on the daughter DNA molecules could serve as a mechanism to regulate replication progression in vivo.