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

Gaggioli V, Le Viet B, Germe T, Hyrien O - Nucleic Acids Res. (2013)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France.

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.

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Time-of-addition-dependent effects of ICRF-193 on nuclear structure and replication foci. (A) Sperm nuclei were incubated in egg extract in the presence of 20 µM rhodamin–dUTP and either drug solvent alone (DMSO) or 100 µM ICRF-193 added at 0 min. Reactions were stopped at the indicated times, fixed and stained with Hoechst. Replication foci were imaged as described in the ‘Materials and Methods’ section. Scale bar = 5 µm. (B) Mean and standard deviation (n = 30) of the number of replication foci per nucleus at 20 min and 30 min with or without ICRF-193. (C) Mean ± SD (n = 50) of nuclear area at 30, 45 and 60 min with DMSO or ICRF-193 added at 0 or 20 min. (D) Replication run-on assay. Sperm nuclei in egg extracts were pulsed for 5 min with rhodamin–dUTP at 40 or 90 min, fixed and stained with Hoechst. (E) Percentage of replicating nuclei at 40 or 90 min in the experiment shown in (D). In all, 150–200 nuclei were counted per sample.
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gkt494-F2: Time-of-addition-dependent effects of ICRF-193 on nuclear structure and replication foci. (A) Sperm nuclei were incubated in egg extract in the presence of 20 µM rhodamin–dUTP and either drug solvent alone (DMSO) or 100 µM ICRF-193 added at 0 min. Reactions were stopped at the indicated times, fixed and stained with Hoechst. Replication foci were imaged as described in the ‘Materials and Methods’ section. Scale bar = 5 µm. (B) Mean and standard deviation (n = 30) of the number of replication foci per nucleus at 20 min and 30 min with or without ICRF-193. (C) Mean ± SD (n = 50) of nuclear area at 30, 45 and 60 min with DMSO or ICRF-193 added at 0 or 20 min. (D) Replication run-on assay. Sperm nuclei in egg extracts were pulsed for 5 min with rhodamin–dUTP at 40 or 90 min, fixed and stained with Hoechst. (E) Percentage of replicating nuclei at 40 or 90 min in the experiment shown in (D). In all, 150–200 nuclei were counted per sample.

Mentions: Sperm nuclei decondensation and replication in the presence or absence of ICRF-193 were monitored by fluorescence microscopy (Figure 2). Sperm nuclei acquired a rounded shape in control extracts but remained elongated when the drug was added at 0 min (Figure 2A). No change in the appearance or mean number of replication foci was observed in the presence of the drug when nuclei were pulse-labeled with rhodamin–dUTP for 2 min at 20 min and 30 min (Figure 2A and B). The mean area of individual nuclei was not significantly affected by ICRF-193 until 30 min but then failed to increase as in the control, showing that the nuclear envelope-dependent decondensation of chromatin was perturbed (Figure 2C), as previously observed (27). Importantly, this perturbation of decondensation was minimized when ICRF-193 was added at 20 min (Figure 2C).Figure 2.


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

Gaggioli V, Le Viet B, Germe T, Hyrien O - Nucleic Acids Res. (2013)

Time-of-addition-dependent effects of ICRF-193 on nuclear structure and replication foci. (A) Sperm nuclei were incubated in egg extract in the presence of 20 µM rhodamin–dUTP and either drug solvent alone (DMSO) or 100 µM ICRF-193 added at 0 min. Reactions were stopped at the indicated times, fixed and stained with Hoechst. Replication foci were imaged as described in the ‘Materials and Methods’ section. Scale bar = 5 µm. (B) Mean and standard deviation (n = 30) of the number of replication foci per nucleus at 20 min and 30 min with or without ICRF-193. (C) Mean ± SD (n = 50) of nuclear area at 30, 45 and 60 min with DMSO or ICRF-193 added at 0 or 20 min. (D) Replication run-on assay. Sperm nuclei in egg extracts were pulsed for 5 min with rhodamin–dUTP at 40 or 90 min, fixed and stained with Hoechst. (E) Percentage of replicating nuclei at 40 or 90 min in the experiment shown in (D). In all, 150–200 nuclei were counted per sample.
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Related In: Results  -  Collection

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gkt494-F2: Time-of-addition-dependent effects of ICRF-193 on nuclear structure and replication foci. (A) Sperm nuclei were incubated in egg extract in the presence of 20 µM rhodamin–dUTP and either drug solvent alone (DMSO) or 100 µM ICRF-193 added at 0 min. Reactions were stopped at the indicated times, fixed and stained with Hoechst. Replication foci were imaged as described in the ‘Materials and Methods’ section. Scale bar = 5 µm. (B) Mean and standard deviation (n = 30) of the number of replication foci per nucleus at 20 min and 30 min with or without ICRF-193. (C) Mean ± SD (n = 50) of nuclear area at 30, 45 and 60 min with DMSO or ICRF-193 added at 0 or 20 min. (D) Replication run-on assay. Sperm nuclei in egg extracts were pulsed for 5 min with rhodamin–dUTP at 40 or 90 min, fixed and stained with Hoechst. (E) Percentage of replicating nuclei at 40 or 90 min in the experiment shown in (D). In all, 150–200 nuclei were counted per sample.
Mentions: Sperm nuclei decondensation and replication in the presence or absence of ICRF-193 were monitored by fluorescence microscopy (Figure 2). Sperm nuclei acquired a rounded shape in control extracts but remained elongated when the drug was added at 0 min (Figure 2A). No change in the appearance or mean number of replication foci was observed in the presence of the drug when nuclei were pulse-labeled with rhodamin–dUTP for 2 min at 20 min and 30 min (Figure 2A and B). The mean area of individual nuclei was not significantly affected by ICRF-193 until 30 min but then failed to increase as in the control, showing that the nuclear envelope-dependent decondensation of chromatin was perturbed (Figure 2C), as previously observed (27). Importantly, this perturbation of decondensation was minimized when ICRF-193 was added at 20 min (Figure 2C).Figure 2.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France.

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.

Show MeSH
Related in: MedlinePlus