Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of topo IIα depletion (A and B) or addition (C and D) on chromatin binding of MCM3, MCM7, RecQ4, Cdc45 and topo IIα. Sperm nuclei were incubated in mock-depleted (A, dotted lines), topo IIα-depleted (A, solid lines) or undepleted (C) extracts added with recombinant htopo IIα (C, dark gray lines) or htopo IIα dilution buffer (C, light gray lines). (A and C) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. The inset shown in (C) illustrates the electrophoretic resolution of human and Xenopus topo IIα. The signals quantified using Image J and normalized to histone H3 are shown as graphs. (B and D) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± S.E.M of MCM signal (normalized to histone H3), in topo IIα-depleted (B) or htopo IIα-added (D) extracts, to MCM signal in control extracts. The observed ratios are significantly (****P < 0.10−4) different from the expected value of 1 in the  hypothesis, as indicated by asterisks and dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753627&req=5

gkt494-F7: Effects of topo IIα depletion (A and B) or addition (C and D) on chromatin binding of MCM3, MCM7, RecQ4, Cdc45 and topo IIα. Sperm nuclei were incubated in mock-depleted (A, dotted lines), topo IIα-depleted (A, solid lines) or undepleted (C) extracts added with recombinant htopo IIα (C, dark gray lines) or htopo IIα dilution buffer (C, light gray lines). (A and C) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. The inset shown in (C) illustrates the electrophoretic resolution of human and Xenopus topo IIα. The signals quantified using Image J and normalized to histone H3 are shown as graphs. (B and D) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± S.E.M of MCM signal (normalized to histone H3), in topo IIα-depleted (B) or htopo IIα-added (D) extracts, to MCM signal in control extracts. The observed ratios are significantly (****P < 0.10−4) different from the expected value of 1 in the hypothesis, as indicated by asterisks and dotted lines.

Mentions: To further understand the role of topoIIα on origin firing time regulation, the binding of topo IIα to sperm chromatin and the effects of topo IIα depletion (Figure 7A and B) or addition (Figure 7C and D) or inhibition (Figure 8) on the chromatin binding of topo IIα, MCM3, MCM7, RecQ4 and Cdc45 proteins were analyzed by western blot. A high concentration of sperm nuclei (5000/µl) was used in these experiments. This improved the detection of chromatin-bound proteins but slowed down replication which started at ∼60 min in undepleted extracts (Supplementary Figure S1) or even later in depleted extracts (not shown).Figure 7.


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)

Effects of topo IIα depletion (A and B) or addition (C and D) on chromatin binding of MCM3, MCM7, RecQ4, Cdc45 and topo IIα. Sperm nuclei were incubated in mock-depleted (A, dotted lines), topo IIα-depleted (A, solid lines) or undepleted (C) extracts added with recombinant htopo IIα (C, dark gray lines) or htopo IIα dilution buffer (C, light gray lines). (A and C) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. The inset shown in (C) illustrates the electrophoretic resolution of human and Xenopus topo IIα. The signals quantified using Image J and normalized to histone H3 are shown as graphs. (B and D) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± S.E.M of MCM signal (normalized to histone H3), in topo IIα-depleted (B) or htopo IIα-added (D) extracts, to MCM signal in control extracts. The observed ratios are significantly (****P < 0.10−4) different from the expected value of 1 in the  hypothesis, as indicated by asterisks and dotted lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753627&req=5

gkt494-F7: Effects of topo IIα depletion (A and B) or addition (C and D) on chromatin binding of MCM3, MCM7, RecQ4, Cdc45 and topo IIα. Sperm nuclei were incubated in mock-depleted (A, dotted lines), topo IIα-depleted (A, solid lines) or undepleted (C) extracts added with recombinant htopo IIα (C, dark gray lines) or htopo IIα dilution buffer (C, light gray lines). (A and C) Purified chromatin at indicated times was analyzed by western blotting for the indicated proteins. Histone H3 was used as a loading control. Samples without egg extract (E-) or without sperm nuclei (S-) and 3 µl of unprocessed extract (E) were loaded as internal controls. The inset shown in (C) illustrates the electrophoretic resolution of human and Xenopus topo IIα. The signals quantified using Image J and normalized to histone H3 are shown as graphs. (B and D) Quantitation of MCM loading over multiple experiments and time points. Shown is the average ratio ± S.E.M of MCM signal (normalized to histone H3), in topo IIα-depleted (B) or htopo IIα-added (D) extracts, to MCM signal in control extracts. The observed ratios are significantly (****P < 0.10−4) different from the expected value of 1 in the hypothesis, as indicated by asterisks and dotted lines.
Mentions: To further understand the role of topoIIα on origin firing time regulation, the binding of topo IIα to sperm chromatin and the effects of topo IIα depletion (Figure 7A and B) or addition (Figure 7C and D) or inhibition (Figure 8) on the chromatin binding of topo IIα, MCM3, MCM7, RecQ4 and Cdc45 proteins were analyzed by western blot. A high concentration of sperm nuclei (5000/µl) was used in these experiments. This improved the detection of chromatin-bound proteins but slowed down replication which started at ∼60 min in undepleted extracts (Supplementary Figure S1) or even later in depleted extracts (not shown).Figure 7.

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