DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts

Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase II alpha (topo II alpha), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo II alpha around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo II alpha depletion accelerated origin cluster activation, and topo II alpha add-back negated overinitiation. Therefore, topo II alpha is not required for DNA replication, but topo II alpha clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo II alpha activity is dispensable for replication and revealing that topo II alpha clamps formed on replicating DNA do not block replication, presumably because topo II alpha acts behind and not in front of forks. Topo II alpha depletion increased, and topo II alpha addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo II alpha restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.