title: DNA topoisomerase IIα 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 IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα 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 IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.
authors.affiliation: Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Gaggioli
authors.forename: Barbara
authors.initials: B
authors.lastname: Le Viet
authors.forename: Thomas
authors.initials: T
authors.lastname: Germe
authors.forename: Olivier
authors.initials: O
authors.lastname: Hyrien
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D059447:Cell Cycle Checkpoints; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D054659:Diketopiperazines; D008297:Male; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D010063:Ovum / Q000166:cytology / Q000201:enzymology*; D010879:Piperazines / Q000494:pharmacology; D018741:Replication Origin; D012093:Replicon; D016196:S Phase; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus / Q000235:genetics* / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D010879:Piperazines; D029867:Xenopus Proteins; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D000251:Adenosine Triphosphatases; C102186:Mcm2 protein, Xenopus; C509836:Mcm7 protein, Xenopus; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D004250:DNA Topoisomerases, Type II