SUMMARYTermination of DNA replication occurs when two replication forks converge upon the same stretch of DNA. Resolution of topological stress by topoisomerases is crucial for fork convergence in bacteria and viruses, but it is unclear whether similar mechanisms operate during vertebrate termination. Using Xenopus egg extracts, we show that topoisomerase II (Top2) resolves topological stress to prevent converging forks from stalling during termination. Under these conditions, stalling arises due to an inability to unwind the final stretch of DNA ahead of each fork. By promoting fork convergence, Top2 facilitates all downstream events of termination. Converging forks ultimately overcome stalling independently of Top2, indicating that additional mechanisms support fork convergence. Top2 acts throughout replication to prevent the accumulation of topological stress that would otherwise stall converging forks. Thus, termination poses evolutionarily conserved topological problems that can be mitigated by careful execution of the earlier stages of replication.

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Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.