Abstract EN:

DNA replication is a complex process that follows an established origin activation program. We demonstrate here that topoisomerase II (topo II) is involved in the establishment of this origin activation program. In interphase xenopus egg extracts, inhibition of topo II by ICRF-193, a drug that traps topo II as closed clamps on DNA, slows down fork progression and delayes origin clusters activation. This was not due to S phase checkpoint activation and only seen if the drug was added during the pre-replicative phase. If added later, during S phase, ICRF-193 does not have effects on DNA replication anymore. Opposite effects of topo II immunodepletion and topo II inhibition indicate that the slower replication with ICRF-193 is not due to topo II catalytic inhibition but to the topo II clamps on DNA that creates obstacles against fork progression. Topo II immunodepletion accelerates S phase by accelerating the activation of origin clusters without altering inter-origin distances. Add-back of recombinant topo II negates these effects. Topo II depletion increases the loading of MCM proteins during origin licensing. Addition of ICRF-193 in G1 does not affect MCM loading. We conclude that i) topo II activity is not required for S phase progression; ii) topo II acts non-catalytically in G1 to cluster origins that undergo reduced MCM loading and thus fire late in S phase; iii) ICRF-193 addition in G1 creates obstacles to origin cluster activation and fork progression, not because of topo II catalytic inhibition, but due to formation of topo II clamps. Topoisomerase II dynamic in G1 phase is important for the determinism of replication origin efficiency