DNA topoisomerase II? controls replication origin cluster licensing and firing time in Xenopus egg extracts.

ABSTRACT: Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase II? (topo II?), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo II? 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? depletion accelerated origin cluster activation, and topo II? add-back negated overinitiation. Therefore, topo II? is not required for DNA replication, but topo II? clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo II? activity is dispensable for replication and revealing that topo II? clamps formed on replicating DNA do not block replication, presumably because topo II? acts behind and not in front of forks. Topo II? depletion increased, and topo II? addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo II? restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.

SUBMITTER: Gaggioli V 

PROVIDER: S-EPMC3753627 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature