Replisomes restrict SMC translocation in vivo.

Structural maintenance of chromosomes (SMC) complexes organize genomes by extruding DNA loops, while replisomes duplicate entire chromosomes. These essential molecular machines must collide frequently in every cell cycle, yet how such collisions are resolved in vivo remains poorly understood. Taking advantage of the ability to load SMC complexes at defined sites in the Bacillus subtilis genome, we engineered head-on and head-to-tail collisions between SMC complexes and the replisome. Replisome progression was monitored by genome-wide marker frequency analysis, and SMC translocation was monitored by time-resolved ChIP-seq and Hi-C. We found that SMC complexes do not impede replisome progression. By contrast, replisomes restrict SMC translocation regardless of collision orientations. Combining experimental data with simulations, we determined that SMC complexes are blocked by the replisome and then released from the chromosome. Occasionally, SMC complexes can bypass the replisome and continue translocating. Our findings establish that the replisome is a barrier to SMC-mediated DNA-loop extrusion in vivo, with implications for processes such as chromosome segregation, DNA repair, and gene regulation that require dynamic chromosome organization in all organisms.