DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts.
Nucleic Acids Res
; 41(15): 7313-31, 2013 Aug.
Article
em En
| MEDLINE
| ID: mdl-23757188
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Óvulo
/
Xenopus
/
Origem de Replicação
/
Proteínas de Ligação a DNA
/
Replicação do DNA
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2013
Tipo de documento:
Article