Spatial organization of supercoil dynamics during DNA replication

Progression of DNA replication inevitably generates helical tension and resulting super-helical structures, or supercoils, that originate from the double helix of DNA. As DNA polymerases progress, negative supercoils accumulate in the wake of the replication fork, thereby impeding fork progression unless the supercoils are resolved topologically. Using super-resolution microscopy combined with spatial distribution analysis, here we describe that SMC5/6-mediated confinement of negative supercoils lies at the basis of releasing them by topoisomerase Top2A. Along with DNA replication, SMC5/6 progressively associates with chromatin and locally increases the density of supercoil clusters behind the fork, allowing Top2A to efficiently target and release the accumulated supercoils. These processes are essential to complete DNA replication prior to mitosis and therefore to ensure genome stability. Remarkably, we found that HeLa cells over-accumulate poorly confined negative supercoils beyond the processing capacity of cellular SMC5/6, which may exemplify the condition associated with genome instability of cancer cells.