TOP1 and TOP2 complementarily maintain DNA replication fork progression in vertebrates

During DNA replication, the topoisomerases TOP1 and TOP2 resolve topological stress associated with DNA unwinding. Based on their catalytic activities, TOP1 is believed to relieve positive supercoil ahead of the replication fork, whereas TOP2 primarily removes topological intertwines between sister chromatids behind the replication fork. As the replication fork rotates, these two TOPs are considered to function complementarily. While this functional interplay between TOP1 and TOP2 has been well established in yeast, it remains unclear whether a similar genetic interaction exists in vertebrate cells. To investigate this, we generated conditionally TOP1-depleted chicken DT40 cells, a model system amenable to gene editing. Although TOP1 is essential in DT40 cells, its depletion did not affect the replication fork progression. Similarly, treatment with ICRF193, a TOP2 inhibitor, had no impact on DNA replication rates. However, the combination of TOP1 depletion and ICRF193 treatment nearly abolished DNA replication, leading to S-phase arrest and rapid apoptosis. Interestingly, treatment of TOP1-depleted cells with etoposide, a TOP2 poison that inhibits DNA religation, did not affect the replication fork progression but instead caused cell cycle arrest in the G1/early S phase, suggesting an impaired initiation of DNA replication. These findings demonstrate that TOP1 and TOP2 have complementary roles in both the progression and initiation of DNA replication in vertebrate cells.